Methods and compositions for detecting target nucleic acids

ABSTRACT

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more ligation probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligation product that is directly detected or that is amplified to produce amplicons that are then detected. The present invention also provides methods to stabilize sample RNA so that degradation does not significantly affect the results of the analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Phase of International ApplicationNo. PCT/US2012/038436 filed May 17, 2012 which claims priority to U.S.Provisional Patent Application No. 61/486,817, filed on May 17, 2011,each of which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No.1R43HG006656-01 awarded by the Small Business Innovation Research (SBIR)program. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The detection of specific nucleic acids is an important tool fordiagnostic medicine and molecular biology research. Gene probe assayscurrently play a role in a number of spheres of diagnostic medicine andmolecular biology, including for example identifying infectiousorganisms such as bacteria and viruses, in probing the expression ofnormal and mutant genes and identifying genes associated with disease orinjury, such as oncogenes, in typing tissue for compatibility precedingtissue transplantation, in matching tissue or blood samples for forensicmedicine, for responding to emergency response situations like a nuclearincident or pandemic flu outbreak, in determining disease prognosis orcausation, and in exploring homology among genes from different species.

Ideally, a gene probe assay should be sensitive, specific and easilyautomatable. The requirement for sensitivity (i.e. low detection limits)has been greatly alleviated by the development of the polymerase chainreaction (PCR) and other amplification technologies which allowresearchers to exponentially amplify a specific nucleic acid sequencebefore analysis. For example, multiplex PCR amplification of SNP lociwith subsequent hybridization to oligonucleotide arrays can be used tosimultaneously genotype hundreds of SNPs.

Specificity is a challenge for gene probe assays. The extent ofmolecular complementarity between probe and target defines thespecificity of the interaction. Variations in composition andconcentrations of probes, targets and salts in the hybridizationreaction as well as the reaction temperature, and length of the probemay all alter the specificity of the probe/target interaction. It can bepossible under some circumstances to distinguish targets with perfectcomplementarity from targets with mismatches, although this is generallydifficult using traditional technology, since small variations in thereaction conditions will alter the hybridization. Techniques formismatch detection include probe digestion assays in which mismatchescreate sites for probe cleavage, and DNA ligation assays where singlepoint mismatches prevent ligation.

A variety of enzymatic and non-enzymatic methods are available fordetecting sequence variations. Examples of enzyme based methods includeInvader™, oligonucleotide ligation assay (OLA) single base extensionmethods, allelic PCR, and competitive probe analysis (e.g. competitivesequencing by hybridization). Enzymatic DNA ligation reactions are wellknown in the art and have been used extensively in SNP detection,enzymatic amplification reactions and DNA repair. A number ofnon-enzymatic or template mediated chemical ligation methods can also beused to detect sequence variations. These include chemical ligationmethods that utilize coupling reagents, such as N-cyanoimidazole,cyanogen bromide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride.

A widely recognized problem when analyzing RNA for genetic studies isthe inherent instability of the RNA itself. RNA naturally has a shortlifetime in living organisms because organisms regulate the RNAconcentration which regulates downstream processes which are dependenton the RNA. There are also many natural processes which lead to thedestruction of an RNA.

In recent years researchers have spent considerable effort developingmethods for the analysis of gene expression in cells. This is generallyaccomplished by analyzing the cell contents for the amount of specificmRNA molecules present. Measurements of gene expression are based on theunderlying assumption that the analyzed RNA sample closely resembles thenumber of transcripts in vivo. Hence, maintaining the integrity of theRNA after extraction from the cell is of paramount importance.Researchers have recognized that transcripts of different genes (mRNA)possess different stabilities which implies that that degradation of RNAoccurring during the isolation procedure may be non-uniformlydistributed among different RNA molecules. One comparison of RNA samplesof different degrees of degradation shows that up to 75% ofmicroarray-based measurements of differential gene expression can becaused by degradation bias. Auer H, Liyanarachchi S, Newsom D, KlisovicM I, Marcucci G, Kornacker K (2003), Nature Genetics 35:292-293.

There remains a need for methods and compositions for efficient andspecific nucleic acid detection and for stabilization of RNA followingextraction from cells.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods and compositions fornon-enzymatic chemical ligation reactions which provides rapid targetdetection and greatly simplified processes of detecting and measuringtarget nucleic acids.

In one aspect, the present invention provides a method for detecting aplurality of different target nucleic acids in a sample, wherein eachtarget nucleic acid comprises a first target domain adjacent to a secondtarget domain and a third target capture domain located upstream ordownstream from the first and second target domains. The methodcomprises the steps of (a) providing a plurality of ligation substrateseach comprising one of the target nucleic acids; a first set of ligationprobes comprising (i) a first nucleic acid ligation probe comprising afirst probe domain hybridized to a first target domain of the one targetnucleic acid sequence and a 5′-ligation moiety; and (ii) a secondnucleic acid ligation probe comprising a second probe domain hybridizedto a second target domain of the one target nucleic acid sequence and a3′ ligation moiety; (b) ligating the first and second ligation probeswithout the use of a ligase enzyme to form a first plurality of ligationproducts; (c) hybridizing target capture probes comprising a capturemoiety to the third target domain of the target nucleic acids to formtarget complexes; (d) capturing the target complexes on a surface usingthe capture moiety; amplifying the ligation products to form amplicons;and (f) detecting the amplicons, thereby detecting the target nucleicacids.

In a further embodiment and in accordance with the above, the targetnucleic acids further comprise a fourth target domain adjacent to afifth target domain and the plurality of ligation substrates eachfurther comprises a second set of ligation probes comprising a thirdnucleic acid ligation probe hybridized to the fourth target domain and afourth nucleic acid ligation probe hybridized to the fifth targetdomain. In such embodiments, the method further comprises the steps ofligating the third and fourth ligation probes in the absence of a ligaseenzyme such that the one target nucleic acid sequence comprises multipleligation products and the detecting step (f) comprises detectingamplicons generated from the multiple ligation products.

In a further embodiment and in accordance with any of the above, thetarget nucleic acids further comprise a sixth target domain adjacent toa seventh target domain, the plurality of ligation substrates eachfurther comprises a third set of ligation probes comprising a fifthnucleic acid ligation probe hybridized to the sixth target domain and asixth nucleic acid ligation probe hybridized to the seventh targetdomain, and the method further comprises ligating the third and fourthligation probes in the absence of a ligase enzyme such that the onetarget nucleic acid sequence comprises multiple ligation products.

In a still further embodiment and in accordance with any of the above,each of the ligation probes further comprises a primer sequence.

In a still further embodiment and in accordance with any of the above,at least one of the ligation probes of each set of ligation probesfurther comprises a variable spacer sequence such that the ampliconshave a target specific length.

In a yet further embodiment and in accordance with any of the above,only one ligation probe of each set of ligation probes comprises thevariable spacer sequence.

In a yet further embodiment and in accordance with any of the above, thevariable spacer sequence is contained between the probe domain and theprimer of the ligation probe.

In a further embodiment and in accordance with any of the above, thecapture moiety of the target capture probe comprises a member selectedfrom: a capture nucleic acid sequence, a bead and a binding partner of abinding partner pair.

In a further embodiment and in accordance with any of the above, the 5′ligation moiety comprises DABSYL and the 3′ ligation moiety comprisesphosphorothioate.

In a further embodiment and in accordance with any of the above, the 3′ligation moiety comprises DABSYL.

In a further embodiment and in accordance with any of the above, thesample was collected into a buffer comprising guanidinium hydrochloride(GuHCl). In an exemplary embodiment, the sample is blood.

In a further embodiment and in accordance with any of the above, thetarget nucleic acids comprise DNA or RNA.

In a still further embodiment and in accordance with any of the above,the detecting step (f) utilizes a technique selected from the groupconsisting of capillary electrophoresis, mass spectrometry, microarrayanalysis, sequencing, real-time PCR, optical detection, fluorescencedetection, bioluminescence detection, chemiluminescence detection,electrochemical detection, electrochemiluminescence detection andlateral flow detection.

In a further embodiment and in accordance with any of the above, thehybridizing step (c) occurs simultaneously with the ligating step (b).

In a further aspect and in accordance with any of the above, the presentinvention provides a method of detecting a plurality of different targetnucleic acids in a sample, wherein each target sequence comprises anadjacent first and a second target domain, the method comprising: (a)providing a plurality of ligation substrates each comprising one of thedifferent target nucleic acids, a first nucleic acid ligation probecomprising a first probe domain complementary to a first target domainof the one target nucleic acid, a first primer sequence; and a5′-ligation moiety; and a second nucleic acid ligation probe comprising:a second probe domain complementary to a second target domain of the onetarget nucleic acid, a second primer sequence, and a 3′ ligation moiety.One of the ligation probe comprises a variable spacer sequence. Themethod further comprises the steps of (b) ligating the first and secondligation probes in the absence of a ligase enzyme to form a plurality ofdifferent ligation products, wherein different ligation products havedifferent target specific lengths; (c) amplifying the ligation product;and (d) detecting the presence of the different ligation products on thebasis of the different target lengths.

In a further embodiment and in accordance with any of the above, thetarget nucleic acid sequences are RNA or DNA.

In a further embodiment and in accordance with any of the above, thesample is derived from blood.

In a still further embodiment and in accordance with any of the above,the sample is derived from paraffin embedded samples.

In a yet further embodiment and in accordance with any of the above, thedetecting is by capillary electrophoresis or by mass spectrometry.

In a further embodiment and in accordance with any of the above, each ofthe first primers are the same and each of the second primers are thesame.

In further aspects and in accordance with any of the above, the presentinvention provides a kit for detecting a target nucleic acid sequence,wherein the target sequence comprises an adjacent first and a secondtarget domain, the kit comprising: (a) 2× lysis buffer comprising 6 MGuHCl; (b) a first ligation probe comprising: (i) a first probe domaincomplementary to the first target domain; (ii) a first primer sequence;and (iii) a 5′-ligation moiety; and (c) a second nucleic acid ligationprobe comprising: (i) a second probe domain complementary the secondtarget domain; (ii) a second primer sequence; and (iii) a 3′ ligationmoiety.

In a further embodiment and in accordance with any of the above, one orboth of the ligation probes further comprise a variable spacer sequence.

In a further aspect and in accordance with any of the above, the presentinvention provides a method of detecting a plurality of different targetnucleic acids in a sample, wherein each target sequence comprises anadjacent first and a second target domain, the method comprising: (a)providing a reaction mixture comprising: (i) a target sample comprisingblood; and (ii) 1× lysis buffer comprising 3 M GuHCl; (b) contacting thereaction mixture with a plurality of different probes sets, each probeset comprising: (i) a first ligation probe comprising: (1) a first probedomain complementary to a first target domain of the one target nucleicacid; (2) a first primer sequence; and (3) a 5′-ligation moiety; and(iii) a second nucleic acid ligation probe comprising: (1) a secondprobe domain complementary to a second target domain of the one targetnucleic acid; (2) a second primer sequence; and (3) a 3′ ligationmoiety; (d) ligating the first and second ligation probes in the absenceof a ligase enzyme to form a plurality of different ligation products;(e) amplifying the different ligation products; and (f) detecting thepresence of the ligation products.

In a further embodiment and in accordance with any of the above, theligation probes further comprises a variable spacer sequence.

In a further embodiment and in accordance with any of the above, thetarget nucleic acids are RNA.

In a further embodiment and in accordance with any of the above, one ofthe first and second ligation probes further comprises one of a bindingpartner pair, and prior to amplifying, a bead comprising the otherbinding pair is added to capture the ligated products.

In a further embodiment and in accordance with any of the above, thedetecting is done using the variable spacer sequence.

In a further aspect and in accordance with any of the above, the presentinvention provides a method for detecting a plurality of target RNAsequences in a sample, where each target nucleic acid sequence comprisesan adjacent first and second target domain and a third target domain.The method includes the steps of collecting a sample into a buffer toform a stabilized sample; conducting an assay in accordance with any ofthe above in the stabilized sample to detect the plurality of targetnucleic acids.

In a further embodiment and in accordance with any of the above, thesample is stable for at least 1 week at ambient room temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of one embodiment of the CLPA-CEassay.

FIG. 2 is a schematic representation of one embodiment of CLPA-MDMassay.

FIG. 3 is a schematic representation showing one embodiment of the2-probe and the 3-probe CLPA reaction.

FIG. 4 is a schematic representation of a DNA synthesis resin that canbe used to manufacture DNA with a 3′-DABSYL leaving group

FIG. 5 is a schematic representation on the process flow for oneembodiment of the CLPA-CE assay

FIG. 6 is a schematic representation showing probe design for a CLPAassay in which the probe contains a size-variant stuffer sequence.

FIG. 7 shows an electrophoretic separation profile on a sample analyzedby CLPA-CE.

FIG. 8 shows the linear relationship between target concentration andpeak height in CLPA-CE analysis.

FIG. 9 shows data for an analysis of FFPE tissue samples comparing aLuminex Signal for various targets with a slice of prostate FFPE tissue(left hand bar for each target) and the no target control (pNTC—righthand bar for each target).

FIG. 10. is a schematic representation of a target capture method usedto separate bound CLPA probe sets from solution phase/unbound CLPA probesets.

FIG. 11 is a schematic illustration of multiple, unique CLPA probe setsthat are bound to the sample target along with a single target captureprobe.

FIG. 12A-C is a further schematic illustration of possible orientationsof one embodiment of the present invention, which can find particularuse in assessing sample integrity. FIG. 12A depicts a similarorientation to FIG. 11, except with the capture probe(s) “downstream” ofthe ligation probe sets. CM is a capture moiety. As will be appreciatedby those in the art, the CM can be on either the 3′ or 5′ terminus ofthe capture probe, although it usually is depicted on the 3′ end. Inaddition, the portion of each ligation probe that does not hybridize toa target domain can contain a number of different functionalities,including, but not limited to, primer binding domains, size tags,capture sequences, etc., as is shown in FIG. 11. FIG. 12A shows asituation where the ligation probe sets are spaced over the length ofthe target in roughly 25-30% increments for a sample integrityassessment. As will be appreciated by those in the art and describedbelow, the spacing of the different ligation probe sets can vary asneeded. FIG. 12B depicts an alternative orientation. FIG. 12C depicts anorientation that can be used both for integrity assessment orredundancy.

FIG. 13A-C depict several schematic of embodiments of the invention foruse in SNP detection.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, phage display, anddetection of hybridization using a label. Specific illustrations ofsuitable techniques can be had by reference to the example herein below.However, other equivalent conventional procedures can, of course, alsobe used. Such conventional techniques and descriptions can be found instandard laboratory manuals such as Genome Analysis: A Laboratory ManualSeries (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: ALaboratory Manual, PCR Primer: A Laboratory Manual, and MolecularCloning: A Laboratory Manual (all from Cold Spring Harbor LaboratoryPress), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York,Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, compositions, formulations andmethodologies which are described in the publication and which might beused in connection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.” It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

I. Overview

The present invention includes methods and compositions for detectingtarget nucleic acids in a sample. In general, target nucleic acids aredetected through methods that include a step of chemical ligation inwhich two ligation probes hybridized to adjacent domains of a targetnucleic acid are ligated without the use of an exogenous ligase enzyme.

The invention provides methods utilizing two or more ligation probes(also referred to herein as “oligonucleotide probes”) that reversiblybind a target nucleic acid in close proximity to each other and possesscomplementary reactive ligation moieties. In the ligation reaction, whenthe probes have bound to the target in the proper orientation, they areable to undergo a spontaneous chemical ligation reaction that yields aligated oligonucleotide product that does not rely on the use of aligase enzyme. The presence of the target(s) of interest can then bedetermined by measuring the presence or amount of ligatedoligonucleotide product (also referred to herein as a “ligationproduct”) in a number of different ways. As is described below, theligation probes can contain a variety of additional functionalities,including, but not limited to, detectable labels to aid in theidentification, quantification or detection of the ligatedoligonucleotide product, including, for example, direct labels such asoptical, and electrochemical labels, etc. as more fully described below,variable spacer sequences or “size tags” comprising nucleic acidsequences that are sized to be specific for a particular target, suchthat detecting ligation products (or amplicons generated from suchligation products) of a particular size identifies the presence and/oramount of a particular target nucleic acid sequence. Another optionalfunctionality for inclusion in one or more of the ligation probes arecapture moieties designed for subsequent capture on a solid support(e.g. microarrays, microbeads, nanoparticles, etc.), which include, butare not limited to, binding partners such as biotin, anchoringoligonucleotide sequences (also referred to herein as “anchor sequences”or “capture sequences”) molecular handles that promote the concentrationor manipulation of the ligated product (magnetic particles,oligonucleotide coding sequences), and promoter and primer sequences tofacilitate subsequent secondary amplification of the ligated product viaan enzyme like a DNA or RNA polymerase.

Preferably, the ligation reactions of the invention do not require thepresence of exogeneously added ligases, nor additional enzymes, althoughsome secondary reactions may rely on the use of enzymes such aspolymerases, as described below. Amplification of the target may alsoinclude turnover of the ligation product, in which the ligation producthas a lower or comparable affinity for the template or target nucleicacid than do the separate ligation probes. Thus, upon ligation of thehybridized probes, the ligation product is released from the target,freeing the target to serve as a template for a new ligation reaction.Alternatively, thermal cycling can be done to remove a ligation productfrom the target sequence and allow new ligation probes to hybridize foranother cycle of ligation.

The invention provides compositions, apparatus and methods for thedetection of one or more nucleic acid targets in a sample including, butnot limited to, DNA and RNA targets. Advantages of using non-enzymaticapproaches for nucleic acid target detection include lower sensitivityto non-natural DNA analog structures, ability to use RNA targetsequences and lower cost and greater robustness under varied conditions.In particular, the methods described herein do not require significantsample preparation; that is, the ligation reactions can be performed inthe presence of contaminants and buffers that would inhibit orinactivate enzymatic processes for detection. For example, blood samplescan be collected into highly denaturing stabilization buffers, theprobes added and the reactions occur, under conditions that woulddenature an enzymatic process. This ability to analyze target nucleicacids, particularly RNA, in impure samples is of particular use inapplications such as medical diagnostics (including gene expressionprofiling and SNP detection), forensic applications, and testing fordamage due to environmental toxins and/or radiation. In addition,methods and compositions of the present invention are useful indetection of nucleic acids from samples that are degraded, includingparaffin-embedded samples in which the process of fixing and embeddingin paraffin resulted in degradation of the samples' nucleic acids.

In addition, one embodiment of the invention provides for assaysrelating to target nucleic acid “integrity”. That is, as is known in theart with mRNA, for example, or nucleic acids in fixed samples, thenucleic acids are degraded over time. As is shown in FIG. 11 and FIG. 12and more fully described below, the present invention allows for the useof multiple ligation complexes to allow for an assessment of theintegrity of the sample. Similarly, the use of these multiple ligationcomplexes per target sequence can also be used for data and assayintegrity through redundancy, similar to running samples in duplicate ortriplicate, for example.

In further aspects, the present invention provides buffers that serve tostabilize nucleic acids in a sample. Such buffers in general include adenaturant comprising a chaotropic cation, including in a non-limitingembodiment, guanidinium hydrochloride. In specific embodiments of theinvention, a sample is collected directly into a buffer of theinvention, and then subsequent hybridization and ligation of ligationprobes is conducted in that buffer without need of purification of thenucleic acids from the sample. In certain embodiments, the samplecollected into the buffer is first diluted and then subsequently methodsdescribed herein of hybridizing and ligating two or more ligation probesare conducted within that diluted sample without need of purification ofthe target nucleic acids in the sample.

As discussed above, ligation probes of the invention are hybridized to atarget nucleic acids and then ligated without the use of a ligaseenzyme. Following ligation, the new product generated (the “ligationproduct”) can optionally be amplified by an enzymatic or chemicalreaction. In the preferred embodiment, the chemical ligation reactionjoins two probes that have PCR primer sites on them, e.g. universal PCRprimers. Additionally, in one embodiment of the invention, one or bothligation probes contain a stuffer sequence, or variable spacer sequence,which is designed to have differing lengths for each probe set (i.e.each target sequence) thereby resulting in a ligation product having atarget-specific length. Following ligation a defined lengtholigonucleotide can now be exponentially amplified by PCR. In accordancewith one aspect of the invention, the probes can possess detectablelabels (e.g. fluorescent labels, electrochemical labels, magnetic beads,nanoparticles, biotin, etc.) to aid in the identification, purification,quantification or detection of the ligated oligonucleotide product. Theprobes may also optionally include in their structure: anchoringoligonucleotide sequences designed for subsequent capture on a solidsupport (microarrays, microbeads, nanoparticles), molecule handles thatpromote the concentration or manipulation of the ligated product(magnetic particles, oligonucleotide coding sequences), and promotersequences to facilitate subsequent secondary amplification of theligated product via an enzyme like a DNA or RNA polymerase.

The ligation reactions of the invention proceed rapidly, are specificfor the target(s) of interest, and can produce multiple copies of theligated product for each target(s), resulting in an amplification(sometimes referred to herein as “product turnover”) of the detectablesignal. The ligation reactions of the invention do not require thepresence of exogeneously added ligases, nor additional enzymes, althoughsome secondary reactions may rely on the use of enzymes such aspolymerases, as described below. Ligation chemistries can be chosen frommany of the previously described chemical moieties. Preferredchemistries are ones that can be easily incorporated into routinemanufacture techniques, are stable during storage, and demonstrate alarge preference for target specific ligation when incorporated into aproperly designed ligation probe set. Additionally, for embodimentswhich involve subsequent amplification by an enzyme, ligationchemistries and probe designs (including unnatural nucleotide analogs)that result in a ligation product that can be efficiently processed byan enzyme are preferred. Amplification of the target may also includeturnover of the ligation product, either by destabilization, e.g. inwhich the ligation product has a lower or comparable affinity for thetemplate or target nucleic acid than do the separate ligation probes, orby standard thermocycling in the presence of excess probes. Thus, uponligation of the hybridized probes, the ligation product is released fromthe target, freeing the target to serve as a template for a new ligationreaction.

In further aspects of the invention and as is discussed in furtherdetail below, specificity of the assays of the invention are optionallyimproved through the use of target capture probes. Target capture probesof the invention include a domain complementary to a domain on thetarget nucleic acid and a capture moiety. The target capture probes donot participate in the ligation reaction with the ligation probes, butare instead designed to hybridize to the target nucleic acid upstream ordownstream from the ligation probes. Hybridization of the target captureprobe to the target nucleic acid produces a target complex that includesthe target nucleic acid, the target capture probe, and any ligationproducts formed on the target nucleic acid. The target complex can thenbe bound to a surface or substrate (such as a bead), and any unboundreactants can be separated from the target complexes bound to thesurface or substrate. Thus, since any subsequent amplification and/ordetection steps are performed on the subset of the original sample oftarget nucleic acids that were successfully hybridized with ligationprobes, the specificity of the subsequent assays is improved.

The above and further aspects and embodiments of the invention aredescribed in further detail in the following sections.

II. Samples

In one aspect, the present invention provides compositions and methodsfor detecting the presence or absence of target nucleic acids (alsoreferred to herein as “target sequences”) in samples. As will beappreciated by those in the art, the samples may comprise any number ofthings, including, but not limited to, bodily fluids (including, but notlimited to, blood, urine, serum, lymph, saliva, anal and vaginalsecretions, perspiration and semen, of virtually any organism, withmammalian samples being preferred and human samples being particularlypreferred); environmental samples (including, but not limited to, air,agricultural, water and soil samples); plant materials; biologicalwarfare agent samples; research samples (for example, the sample may bethe product of an amplification reaction, for example generalamplification of genomic DNA); purified samples, such as purifiedgenomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomicDNA, etc.); as will be appreciated by those in the art, virtually anyexperimental manipulation may have been done on the sample. Someembodiments utilize siRNA and microRNA as target sequences (Zhang etal., J Cell Physiol. (2007) 210(2):279-89; Osada et al., Carcinogenesis.(2007) 28(1):2-12; and Mattes et al., Am J Respir Cell Mol. Biol. (2007)36(1):8-12, each of which is incorporated herein by reference in itsentirety for all purposes, and in particular all teachings related totarget sequences).

Some embodiments of the invention utilize samples from stored (e.g.frozen and/or archived) or fresh tissues. Paraffin-embedded samples areof particular use in many embodiments, as these samples can be veryuseful for diagnosis and prognosis, due to the presence of additionaldata associated with the samples. Fixed and paraffin-embedded tissuesamples as described herein refers to storable or archival tissuesamples. Most patient-derived pathological samples are routinely fixedand paraffin-embedded to allow for histological analysis and subsequentarchival storage. Such samples are often not useful for traditionalmethods of nucleic acid detection, because such studies require a highintegrity of the nucleic acid sample so that an accurate measure ofnucleic acid expression can be made. Often, gene expression studies inparaffin-embedded samples are limited to qualitative monitoring by usingimmunohistochemical staining to monitor protein expression levels.

A number of techniques exist for the purification of nucleic acids fromfixed paraffin-embedded samples as described in WO 2007/133703 theentire contents of which is herein incorporated by reference for allpurposes and in particular for all teachings related to the purificationof nucleic acids from paraffin-embedded samples. Methods described byFoss, et al Diagnostic Molecular Pathology, (1994) 3:148-155 and Paska,C., et al Diagnostic Molecular Pathology, (2004) 13:234-240 as well ascommercially available kits like Ambion's Recoverall Total Nucleic acidIsolation kit are included by reference in their entirety. Commonmethods start with a step that removes the paraffin from the tissue viaextraction with Xylene or other organic solvent, followed by treatmentwith heat and a protease like proteinase K which cleaves the tissue andproteins and helps to release the genomic material from the tissue. Thereleased nucleic acids are then captured on a membrane or precipitatedfrom solution, washed to removed impurities and for the case of mRNAisolation, a DNase treatment step is sometimes added to degrade unwantedDNA.

As will be discussed in further detail herein, both the target analytesand the ligation probes used to detect the target analytes may inaccordance with the invention comprise nucleic acids. By “nucleic acid”or “oligonucleotide” or grammatical equivalents herein means at leasttwo nucleotides covalently linked together. The target nucleic acids maycomprise DNA or RNA. A nucleic acid of the present invention willgenerally contain phosphodiester bonds (for example in the case of thetarget sequences), although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones(particularly for use with the ligation, label or capture probes),comprising, for example, phosphoramide (Beaucage et al., Tetrahedron(1993) 49(10):1925 and references therein; Letsinger, J. Org. Chem.(1970) 35:3800; Sprinzl et al., Eur. J. Biochem. (1977) 81:579;Letsinger et al., Nucl. Acids Res. (1986) 14:3487; Sawai et al, Chem.Lett. (1984) 805; Letsinger et al., J. Am. Chem. Soc. (1988) 110:4470;and Pauwels et al., Chemica Scripta (1986) 26:141), phosphorothioate(Mag et al., Nucleic Acids Res. (1991) 19:1437; and U.S. Pat. No.5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. (1989)111:2321, 0-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. (1992) 114:1895; Meier et al., Chem. Int. Ed. Engl.(1992) 31:1008; Nielsen, Nature, (1993) 365:566; Carlsson et al., Nature(1996) 380:207, all of which are incorporated herein by reference intheir entirety). Other analog nucleic acids include those with bicyclicstructures including locked nucleic acids, Koshkin et al., J. Am. Chem.Soc. (1998) 120:13252 3); positive backbones (Denpcy et al., Proc. Natl.Acad. Sci. USA (1995) 92:6097; non-ionic backbones (U.S. Pat. Nos.5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi etal., Angew. Chem. Intl. Ed. English (1991) 30:423; Letsinger et al., J.Am. Chem. Soc. (1988) 110:4470; Letsinger et al., Nucleoside &Nucleotide (1994) 13:1597; Chapters 2 and 3, ASC Symposium Series 580,Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic &Medicinal Chem. Lett. (1994) 4:395; Jeffs et al., J. Biomolecular NMR(1994) 34:17; Xu et al., Tetrahedron Lett. (1996) 37:743) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. All of these references are herein expressly incorporated byreference in their entirety for all purposes, and in particular for allteachings related to nucleic acids. These modifications of theribose-phosphate backbone may be done to facilitate the addition oflabels or other moieties, to increase or decrease the stability andhalf-life of such molecules in physiological environments, etc.

For example, the use of the ligation moieties of the invention can, insome cases depending on the chemistry utilized, result in nucleic acidanalogs as the ligation product. For example, as shown in Scheme 1,below, the use of certain ligation moieties result in a phosphothioesterbonds.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of a ligation moiety, an analog structure may be used.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Nucleic acid analogue may include, for example, peptide nucleic acid(PNA, WO 92/20702, incorporated herein by reference in its entirety) andLocked Nucleic Acid (LNA, Koshkin A A et al. Tetrahedron (1998)54:3607-3630, Koshkin A A et al. J. Am. Chem. Soc. (1998)120:13252-13253, Wahlestedt C et al. PNAS (2000) 97:5633-5638, each ofwhich is incorporated herein by reference in its entirety). In someapplications analogue backbones of this type may exhibit improvedhybridization kinetics, improved thermal stability and improvedsensitivity to mismatch sequences.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequences. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includingnaturally occurring nucleobases (uracil, adenine, thymine, cytosine,guanine) and non-naturally occurring nucleobases (inosine, xathaninehypoxathanine, isocytosine, isoguanine, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). 5-propynyl-uracil, 2-thio-5-propynyl-uracil)etc. As used herein, the term “nucleobase” includes both “nucleosides”and “nucleotides”, and monomers of nucleic acid analogs. Thus, forexample, the individual units of a peptide nucleic acid, each containinga base, are referred to herein as a nucleobase.

Nucleic acid samples (e.g. target sequences) that do not exist in asingle-stranded state in the region of the target sequence(s) aregenerally rendered single-stranded in such region(s) prior to detectionor hybridization. Generally, nucleic acid samples can be renderedsingle-stranded in the region of the target sequence using heat orchemical denaturation. For polynucleotides obtained via amplification,methods suitable for generating single-stranded amplification productsare preferred. Non-limiting examples of amplification processes suitablefor generating single-stranded amplification product polynucleotidesinclude, but are not limited to, T7 RNA polymerase run-offtranscription, RCA, Asymmetric PCR (Bachmann et al., Nucleic Acid Res.(1990) 18:1309), and Asynchronous PCR (WO 01/94638). Commonly knownmethods for rendering regions of double-stranded polynucleotides singlestranded, such as the use of PNA openers (U.S. Pat. No. 6,265,166), mayalso be used to generate single-stranded target sequences on apolynucleotide.

Target Nucleic Acids

As discussed further herein, the invention provides methods andcompositions for detecting target sequences. By “target sequence” or“target nucleic acid” or grammatical equivalents herein means a nucleicacid sequence on a single strand of nucleic acid. The target sequencemay be a portion of a gene, a regulatory sequence, genomic DNA, cDNA,RNA including mRNA, MicroRNA and rRNA, or others. As is outlined herein,the target sequence may be a target sequence from a sample, or asecondary target such as a product of an amplification reaction, etc. Itmay be any length, with the understanding that longer sequences are morespecific. As will be appreciated by those in the art, the complementarytarget sequence may take many forms. For example, it may be containedwithin a larger nucleic acid sequence, i.e. all or part of a gene ormRNA, a restriction fragment of a plasmid or genomic DNA, among others.Any and all combinations of these may serve as target nucleic acids in aparticular assay. In many cases, multiplex assays are done, where aplurality of target sequences are simultaneously detected, such as forgene expression profiling as is more fully described below.

In general, each target sequence is comprised of a plurality ofdifferent target domains. Each target sequence has at least a pair ofligation domains for hybridization to a set of ligation probes, or more,as described below. For example, a first target domain of a sampletarget sequence may hybridize to a first ligation probe, and a secondtarget domain in the target sequence may hybridize to a second ligationprobe, such as to bring the chemical ligation moieties into spatialproximity sufficient to allow spontaneous chemical ligation.

In general, each pair of target ligation domains is adjacent to eachother, that is, there are no nucleotides separating the two domains.This finds use in both general detection of target sequences (e.g. geneexpression profiling using mRNA as the target sequences), transferreactions as discussed below, as well as for single nucleotidepolymorphism (SNP) detection. For SNP detection, the target sequencecomprises a position for which sequence information is desired,generally referred to herein as the “detection position”. In someembodiments, the detection position is a single nucleotide, although insome embodiments, it may comprise a plurality of nucleotides, eithercontiguous with each other or separated by one or more nucleotides. By“plurality” as used herein is meant at least two. As used herein, thebase of a ligation probe which basepairs with the detection positionbase in a hybrid is termed the “interrogation position”.

Each sample target nucleic acid can additionally have multiple pairs ofligation domains. That is, 1, 2, 3 or more sets of ligation probes canhybridize to the same target sequence at multiple locations, as isgenerally depicted in FIG. 11 or 12. As is more fully outlined below,the use of multiple ligation domains per target nucleic acid can serveas the basis to assess the integrity of the target nucleic acids (and/orthe original sample) in the sample.

The sample target nucleic acids may contain other domains, in additionto ligation domains. In certain embodiments, the target nucleic acids ofthe invention include a target capture domain to which a target capturedomain is able to hybridize. In general, as depicted in FIG. 11 anddepending on the purpose of the assay, a target capture domain can be“upstream”, “downstream” or “in-between” one or more of the ligationdomains of the target nucleic acid.

Unless specified, the terms “first” and “second” are not meant to conferan orientation of the sequences with respect to the 5′-3′ orientation ofthe target sequence. For example, assuming a 5′-3′ orientation of thecomplementary target sequence, the first target domain may be locatedeither 5′ to the second domain, or 3′ to the second domain. For ease ofreference and not to be limiting, these domains are sometimes referredto as “upstream” and “downstream”, with the normal convention being thetarget sequence being displayed in a 5′ to 3′ orientation. However, itshould be noted that ligation domains have an orientation such that the3′ and 5′ ligation moieties of the ligation probe sets hybridize eithercompletely adjacently (e.g. no intervening nucleobases) or within adistance that the linkers attaching the ligation moieties allow forligation.

In some embodiments, the pair of target ligation domains may beseparated. For example, in some cases, when ligation amplification isdesired, the ligation probes may utilize linkers and be separated whenhybridized by one or more nucleobases of the target sequence to conferhybridization instability on the ligated product. In other applications,As will be discussed in further detail below, buffers of the inventionare of use in stabilizing nucleic acids, particularly RNA, in a sample.In some aspects of the invention, samples are collected into buffers ofthe invention. In further embodiments, and as is discussed in furtherdetail below, such buffers include one or more of a denaturant, areducing agent, a surfactant, a pH buffer, EDTA, and any combinationthereof.

III. Buffers

In one aspect, the invention provides methods and compositions whichstabilize nucleic acids (also referred to herein as “sample nucleicacid” or “target nucleic acids”). By “stabilize” as used herein is meantthat the nucleic acids in a sample are resistant to degradation evenwhen stored at ambient room temperature or above for a period of time.In some embodiments, nucleic acids contained in buffers of the inventionare stable at room temperature or above for about one day to about threemonths. Stability can be measured using any means known in the art,including assays for nucleic acid integrity as further discussed below.In further embodiments, a sample comprising nucleic acids contained in abuffer of the invention is assessed as having increased stability ascompared to a sample that was not stored in the buffer if at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the nucleic acids in thesample stored in the buffer show less degradation than those in thesample that was not stored in the buffer. In yet further embodiments, asample is identified as being stabilized by the buffers of the presentinvention if at least a majority of the nucleic acids in the sample showreduced degradation as compared to a sample that was not stored in thebuffer. Stability of RNA samples are often assessed by CapillaryElectrophoresis methodologies that look to measure the average size ofthe nucleic acid sample. Stabilized samples will have a longer averagesize than non-stabilized samples. Another aspect of this invention isthe use of multiple ligation probe sets combined with one or more targetcapture probes that can be used to assess the average size of a targetnucleic acid, and by correlation, the level of degradation of the targetnucleic acid.

Buffers of the invention can optionally and in any combination includeone or more of a denaturant, a reducing agent, a surfactant, a pHbuffer, a chelator such as EDTA, and any combination thereof. As will beappreciated, buffers of the invention may include multiple types ofcomponents within the same class—e.g., buffers of the invention mayinclude one or more different kinds of denaturants in combination withone or more types of surfactants, and so on.

An advantage of the buffers of the present invention is that they can beused to stabilize nucleic acids such as RNA in a sample and then thesample can be directly analyzed from the buffer solutions in accordancewith the methods described herein. In other words, samples contained inbuffer solutions of the invention can be subjected to the chemicalligation and detection methods described herein without isolation orpurification of the RNA. Another advantage of the buffers of theinvention is that cell lysis occurs upon the collection of the sample inthe buffer, thus not requiring an additional lysis step to release thetarget nucleic acids from the sample.

In an exemplary embodiment, a sample comprising RNA can be combined in abuffer solution comprising guanidinium hydrochloride,ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), TritonX-100, and Tris-HCL at a pH of 7.5. In another embodiment, the samplecomprising RNA can be combined in a buffer solution comprisingguanidinium isothiocyanate, EDTA, DTT, Triton X-100, and Tris-HCl at apH of 7.5. The RNA is stable in such buffer solutions and it is notnecessary to isolate the RNA from other sample constituents which mayenhance degradation of the RNA.

In further embodiments, the buffers of the invention preferably includea denaturant, particularly a chaotropic cation, that has the effect ofincreasing reaction and binding efficiency in the methods and assaysdescribed herein by helping to unfold the secondary structure of theRNA. Common Chaotropic molecules are guanidinium hydrochloride,guanidinium isothiocyanate, betaine or glycine betaine, urea, thiourea,and lithium perchlorate. Without being bound by theory, chaotropicagents that are effective in breaking of tertiary structure in nucleicacids are preferred and chaotropic agents that also maintain thesolubility of the nucleic acid target in solution are particularlybeneficial. An advantage of buffers of the invention, particularlybuffers comprising a chaotropic cation, is that the buffer keeps thenucleic acids of the sample in solution. This is in contrast to othertraditional buffers used in transport systems for blood-based tests,which tend to precipitate/form a cationic shell around the nucleic acidsof the sample (particularly RNA). Since the buffers of the inventionkeep the nucleic acids in solution, and since the chemical ligationmethods of the assays of the invention do not require enzymes, a samplecan be collected into a buffer and the ligation probes (and in manyembodiments, target capture probes) can be added to the sample andligation products formed. To change hybridization conditions to thenrelease the ligation products or target complexes for further analysis,the sample plus buffer can simply be diluted to dilute the denaturantand thereby change the hybridization conditions, thus allowing analysisof the nucleic acids using any of the methods described herein and knownin the art.

In further embodiments, the buffers of the invention have a pH of about5 to about 8.5. More preferably the buffer solution has a pH of about 6to 8 and even more preferably, a pH of approximately 7.3 or 7.5.

The following sections discuss exemplary buffer components in furtherdetail. Although each of these components is discussed separately, thepresent invention encompasses any combination of the following buffercomponents as well as any other components known in the art.

Denaturants

In preferred embodiments, buffers of the present invention include oneor more denaturants. By denaturant as used herein is meant any substancethat serves to unfold the double helix of nucleic acids with loss ofsecondary and tertiary structure. In further embodiments, thedenaturants comprise a chaotropic cation, including without limitationguanidinium hydrochloride (GuHCl) and guanidinium isothiocyanate.

In further embodiments, the denaturant is guanidinium hydrochloride,which is present in a concentration from about 1 molar to about 8 molarand more preferably, a concentration of about 2 molar to about 4 molar,and even more preferably, a concentration of approximately 3 molar. Infurther embodiments, concentration of GuHCl in buffers of the inventionrange from about 0.2-10, 0.5-9, 1-8, 1.5-7, 2-6, 2.5-5, and 3.0-4.0molar. In still further embodiments, concentrations of GuHCl in buffersof the invention are about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 molar.

In other embodiments, the denaturant is guanidinium isothiocyanate,which is present in a concentration from about 1 molar to about 8 molarand more preferably, a concentration of about 2 molar to about 4 molar,and even more preferably, a concentration of approximately 3 molar. Infurther embodiments, concentration of guanidinium isothiocyanate inbuffers of the invention range from about 0.2-10, 0.5-9, 1-8, 1.5-7,2-6, 2.5-5, and 3.0-4.0 molar. In still further embodiments,concentrations of guanidinium isothiocyanate in buffers of the inventionare about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15molar.

As will be appreciated, other denaturants known in the art can be usedin buffers of the invention at similar concentrations as those listedabove for guanidinium hydrochloride and guanidinium isothiocyanate.

In some embodiments, such as with the use of high concentrations ofsalts such as guanidinium salts, these reagents also serve as lysisagents. As will be appreciated by those in the art, in general, the useof a denaturant that also serves as a cell lysis agent is of particularuse, although the present invention also contemplates the use of a firstseparate lysis step followed by the addition of the denaturant.

Surfactants

In some embodiments, buffers of the present invention include one ormore surfactants. In further embodiments, the surfactant includeswithout limitation Triton X-100 and sodium N-lauroylsarcosine.

In further embodiments, the surfactant is present in buffers of theinvention at a concentration from about 0.1% to about 5% by weight. Instill further embodiments, the surfactant is present in a concentrationof about 0.1%-10%, 0.5%-9.5%, 1%-9%, 1.5%-8.5%, 2%-8%, 2.5%-7.5%, 3%-7%,3.5%-6.5%, 4%-6%, and 4.5%-5.5% by weight. In preferred embodiments, thesurfactant has a concentration of about 0.5% to about 3%. In a furtherembodiment, the surfactant has a concentration of approximately 1.5% byweight.

ph Buffer

In some embodiments, buffers of the present invention include one ormore pH buffers. Such pH buffers include without limitation Tris. Inother embodiments the pH buffer can be one of many known by thoseskilled in the art. Generally the pH buffer used in the presentinvention includes an agent that has a pKa within one pH unit of theoperating pH.

In some embodiments, the pH buffer is present in buffers of theinvention at a concentration from about 10 mM to about 100 mM. Inpreferred embodiments, the pH buffer has a concentration of about 20 mMto about 50 mM and more preferably, a concentration of approximately 30mM. In further embodiments, the pH buffer has a concentration of about5-150, 10-140, 15-130, 20-120, 25-110, 30-100, 35-90, 40-80, 45-70, and50-60 mM.

Reducing Agents

In some embodiments, buffers of the present invention include one ormore reducing agents. Such reducing agents can include withoutlimitation Dithiothreitol (DTT) and mercaptoethanol.

In further embodiments, the reducing agents have a concentration fromabout 1 mM to about 100 mM. In preferred embodiments, the reducing agenthas a concentration of about 4 mM to about 7 mM and even morepreferably, a concentration of approximately 5 mM. In still furtherembodiments, the reducing agents have a concentration of about 0.5-10,1-9.5, 1.5-9, 2-8.5, 2.5-8, 3-7.5, 3.5-7, 4-6.5 mM. In yet furtherembodiments, the reducing agents have a concentration of about 1-150,10-140, 15-130, 20-120, 25-110, 30-100, 35-90, 40-80, 45-70, and 50-60mM.

EDTA

In further embodiments, buffers of the invention include EDTA at aconcentration of from about 1 mM to about 100 mM. More preferably theEDTA has a concentration of about 10 mM to about 50 mM and even morepreferably, a concentration of approximately 20 mM. In furtherembodiments, the EDTA is present at a concentration of about 1-150,10-140, 15-130, 20-120, 25-110, 30-100, 35-90, 40-80, 45-70, and 50-60mM. In still further embodiments, the EDTA has a concentration of about5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mM.

Additional Buffer Components

The buffers of the invention may further include any additionalcomponents known in the art, particularly components known in the art tobe of use in reactions involving nucleic acids. Additional componentsmay include without limitation: adjuvants, diluents, binders,stabilizers, salts (including NaCl and MgCl₂), lipophilic solvents,preservatives, or the like. Buffer components may also includepharmaceutical excipients and additives, proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. Carbohydrate excipients are also intendedwithin the scope of this invention, examples of which include but arenot limited to monosaccharides such as fructose, maltose, galactose,glucose, D-mannose, sorbose, and the like; disaccharides, such aslactose, sucrose, trehalose, cellobiose, and the like; polysaccharides,such as raffinose, melezitose, maltodextrins, dextrans, starches, andthe like; and alditols, such as mannitol, xylitol, maltitol, lactitol,xylitol sorbitol (glucitol) and myoinositol.

Uses of Buffers of the Invention

Buffers of the invention can be used with any of the sample collection,chemical ligation, or assays discussed herein.

In some embodiments, samples containing target nucleic acids arecollected into buffers of the invention. The advantage of buffers of theinvention is that they tend to stabilize nucleic acids contained in thesamples. Thus, degradation that often begins to occur, particularly withRNA, immediately upon collection is prevented to at least some degree bythe buffers of the invention, providing the advantage of a robust set oftarget nucleic acids for further analysis using methods of theinvention.

In further embodiments, upon collection of samples into buffers of theinvention, ligation probes and optionally target capture probes (whichare further discussed in detail below) can be added directly to thesample in the buffer without purification of the target nucleic acids.In some embodiments, the sample in the buffer is first diluted prior tothe addition of the probes (including ligation and target captureprobes). Because the ligation methods of the present invention are notreliant on enzymes, hybridization and ligation of ligation probes canoccur without purifying the nucleic acids from the sample and withoutrelying on the use of a ligase enzyme. This further serves to limit thedegradation that occurs in the nucleic acids of the sample.

IV. Ligation Probes and Chemical Ligation Methods

In one aspect, ligation probes of the invention comprise any polymericspecies that is capable of interacting with a nucleic acid target(s) ina sequence specific manner and possess chemical moieties allowing theprobes to undergo a spontaneous chemical ligation reaction with anotherpolymeric species possessing complementary chemical moieties. In oneembodiment, the ligation probes can be DNA, RNA, PNA, LNA, modifiedversions of the aforementioned and/or any hybrids of the same (e.g.DNA/RNA hybrids, DNA/LNA hybrids, DNA/PNA hybrids). In a preferredembodiment, the ligation probes comprise DNA or RNA oligonucleotides.

Ligation probes of the invention are designed such that when the probesbind to a part of the target polynucleotide in close spatial proximity,a chemical ligation reaction occurs between the probes. In general, theprobes comprise chemically reactive moieties (herein generally referredto as “ligation moieties”) and bind to the target polynucleotide in aparticular orientation, such that the chemically reactive moieties comeinto close spatial proximity, thus resulting in a spontaneous ligationreaction that can take place without the use of a ligase enzyme.

In one embodiment, the invention provides sets of ligation probes,usually a first and a second ligation probe, although as is describedherein some embodiments utilize more than two. In addition, as notedherein, in some cases a transfer reaction is done rather than ligation;“ligation probes” includes “transfer probes”. Each ligation probecomprises a nucleic acid portion, sometimes referred to herein as a“ligation domain” or “probe domain” that is substantially complementaryto one of the target domains. Probes of the present invention aredesigned to be complementary to a target sequence such thathybridization of the target sequence and the probes of the presentinvention occurs. As outlined herein, this complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the probesof the present invention. However, if the number of mutations is sogreat that no hybridization can occur under even the least stringent ofhybridization conditions, the sequence is not a complementary sequence.Thus, by “substantially complementary” herein is meant that the probesare sufficiently complementary to the target sequences to hybridizeunder normal reaction conditions. “Identical” sequences are those thatover the length of the shorter sequence of nucleobases, perfectcomplementarity exists. A variety of hybridization conditions may beused in the present invention, including high, moderate and lowstringency conditions; see for example Maniatis et al., MolecularCloning: A Laboratory Manual, 2d Edition, 1989, and Ausubel, et al,Short Protocols in Molecular Biology, herein incorporated by reference.The hybridization conditions may also vary when a non-ionic backbone,e.g. PNA is used, as is known in the art.

In one aspect of the invention, the length of the probe is designed tovary with the length of the target sequence, the specificity required,the reaction (e.g. ligation or transfer) and the hybridization and washconditions. Generally, in this aspect ligation probes range from about 5to about 150 nucleobases, with from about 15 to about 100 beingpreferred and from about 25 to about 75 being especially preferred. Ingeneral, these lengths apply equally to ligation and transfer probes.

In another embodiment of the invention, referred to herein as “CLPA-CE”which is described more fully below, probe length is designed to varyfor each target of interest thereby generating ligation products thatcan be identified and analyzed based on length variance.

Ligation probes of the invention are designed to be specific for thepolynucleotide target. These probes bind to the target in close spatialproximity to each other and are oriented in such a manner that thechemically reactive ligation moieties are in close spatial proximity. Inone aspect, two or more probes are designed to bind near adjacent siteson a target polynucleotide. In a preferred embodiment, two probes bindto the target such that the ligation moiety at the 5′ end of oneoligonucleotide probe is able to interact with the ligation moiety atthe 3′ end of the other probe. In preferred embodiments, the ligationreaction between the ligation moieties occurs without the use of anexogenous ligase enzyme, also referred to herein as “chemical ligation.”It should be noted that the sets of ligation probes do not ligate usingligases due to the presence of the ligation moieties.

In the case of SNP detection, a set of ligation probes may in some casesactually comprise be three or four or five different ligation probes,some of which are allele specific. By “allele specific” probe or primeris meant a probe or primer that hybridizes to a target sequence anddiscriminates between alleles. In this case, for example, when a SNP isbiallelic (such as depicted in FIG. 13), a set of three ligation probesare used, two of which contain a different nucleotide at the detectionposition. That is, as is known in the art, mismatches at the junction ofa ligation complex will either result in no ligation or a decreasedamount of ligation. So, depending on the orientation of the probes, thedetection position can either be on the terminal position of the“upstream” probe or the downstream probe as is depicted in FIGS. 13A and13C). The mismatch can also be positioned internally to a probe and themismatch discrimination is based on differences in the binding strengthor Tm of the mismatch discriminating probes (as depicted in FIG. 13B. Inthe case of a biallelic SNP, two of the probes have the same probedomain except that the nucleotide at the detection position,corresponding to the interrogation position on the target sequence, isdifferent. Thus, depending on whether the patient is homozygotic (T/T orC/C) or heterozygotic (T/C or C/T), the ligation between probes occurs.Furthermore, in this invention, there is generally an additionaldifference between the mismatch detection ligation probes that enablesthe easy identification of which probe was preferentially ligated. Forexample, the “A” allelic probe may have a variable spacer sequence of 15nucleotides (15 mer) and the “G” allelic probe may have a variablespacer sequence of 20 nucleotides (20 mer), the probes may havedifferent capture domains or label sequences, etc.

Similarly, as will be appreciated by those in the art, triallelic orquadallelic SNPs use 4 (3 probes with the same target domain and onewith the other target domain) or 5 (4 probes with the same target domainand one with the other target domain) ligation probes in the set.

The size of the primer and probe nucleic acid may vary, as will beappreciated by those in the art with each portion of the probe and thetotal length of the probe in general varying from 5 to 500 nucleotidesin length. Each portion is preferably between 10 and 100 beingpreferred, between 15 and 50 being particularly preferred, and from 10to 35 being especially preferred, depending on the use and amplificationtechnique. Thus, for example, the universal priming site(s) of theprobes are each preferably about 15-20 nucleotides in length, with 18being especially preferred. The adapter sequences of the probes arepreferably from 15-25 nucleotides in length, with 20 being especiallypreferred. The target specific portion of the probe is preferably from15-50 nucleotides in length. In addition, the primer may include anadditional amplification priming site. In a preferred embodiment theadditional amplification priming site is a T7 RNA polymerase primingsite.

A number of non-enzymatic or template mediated chemical ligation methodscan be used in accordance with the present invention. These includechemical ligation methods that utilize coupling reagents, such asN-cyanoimidazole, cyanogen bromide, and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. SeeMetelev, V. G., et al., Nucleosides & Nucleotides (1999) 18:2711;Luebke, K. J., and Dervan, P. B. J. Am. Chem. Soc. (1989) 111:8733; andShabarova, Z. A., et al., Nucleic Acids Research (1991) 19:4247, each ofwhich is incorporated herein by reference in its entirety for allpurposes and in particular for all teachings related to chemicalligation. Kool (U.S. Pat. No. 7,033,753), which is incorporated hereinby reference in its entirety, describes the use of chemical ligation andfluorescence resonance energy transfer (FRET) to detect geneticpolymorphisms. The readout in this process is based on the solutionphase change in fluorescent intensity. Terbrueggen (US PatentPublication No. 12008/0124810) which is incorporated herein by referencein its entirety describes the use of chemical ligation methods,compositions and reagents for the detection of nucleic acids viamicroarray detection. Other chemical ligation methods react a5′-tosylate or 5′-iodo group with a 3′-phosphorothioate group, resultingin a DNA structure with a sulfur atom replacing one of the bridgingphosphodiester oxygen atoms. See Gryanov, S. M., and Letsinger, R. L.,Nucleic Acids Research (1993) 21:1403; Xu, Y. and Kool, E. T.Tetrahedron Letters (1997) 38:5595; and Xu, Y. and Kool, E. T., NucleicAcids Research (1999) 27:875, each of which is herein incorporated byreference in its entirety. Letsinger et al (U.S. Pat. No. 5,780,613,herein incorporated by reference in its entirety) have previouslydescribed an irreversible, nonenzymatic, covalent autoligation ofadjacent, template-bound oligonucleotides wherein one oligonucleotidehas a 5′ displaceable group and the other oligonucleotide has a 3′thiophosphoryl group. Each of these references describing chemicalligation methods is incorporated herein by reference in its entirety forall purposes and in particular for all teachings related to chemicalligation.

In one aspect, the ligation reactions of the invention include transferreactions. In this embodiment, the probes hybridize to the targetsequence, but rather than oligonucleotide probes being ligated togetherto form a ligation product, a nucleic acid-directed transfer of amolecular entity (including reporter molecules such as fluorophores,quenchers, etc) from one oligonucleotide probe to other occurs. Thistransfer reaction is analogous to a ligation reaction, however insteadof joining of two or more probes, one of the probes is ligated to thetransfer molecule and the other probe is the “leaving” of the chemicalreaction. Importantly, similar to the ligation reaction, the transferreaction is facilitated by the proximal binding of the transfer probesonto a nucleic acid target, such that significant signal is detectedonly if the probes have hybridized to the target nucleic acid in closeenough proximity to one another (e.g., at adjacent sites) for thetransfer reaction to take place.

In one aspect, the invention relates to methods of chemical ligationthat include the binding of at least a first and a second ligation probeto the target nucleic acid to form a “ligation substrate” underconditions such that the ligation moieties of the first and secondligation probes are able to spontaneously react, ligating the probestogether, in the absence of exogenous ligase; that is, no exogenousligase is added to the reaction and instead the reaction proceedswithout the use of a ligase. In the case of the transfer reaction, thismay be referred to as either a “ligation substrate” or a “transfersubstrate”. By “ligation substrate” herein is meant a substrate forchemical ligation comprising at least one target nucleic acid sequenceand two or more ligation probes. Similarly, included within thedefinition of “ligation substrate” is a “transfer substrate”, comprisingat least one target nucleic acid sequence and two or more transferprobes. Once the chemical ligation step has occurred, the product ofreaction is sometimes referred to as a “ligation complex”, comprisingthe ligated probes and the target to which they are still hybridized.

In some embodiments of the invention, for example when additionalspecificity is desired, more than two ligation probes can be used, as isgenerally depicted in FIG. 3. In this embodiment, the “middle” ligationprobe(s) can also be adjacent or separated by one or more nucleobases ofthe target sequence. In a preferred embodiment, the ligation reactiondoes not require the presence of a ligase enzyme and occursspontaneously between the bound probes in the absence of any addition(e.g. exogeneous) ligase.

Chemical ligation can, under appropriate conditions, occur spontaneouslywithout the addition of any additional activating reagents or stimuli.Alternatively, “activating” agents or external stimuli can be used topromote the chemical ligation reaction. Examples of activating agentsinclude, without limitation, carbodiimide, cyanogen bromide (BrCN),imidazole, 1-methylimidazole/carbodiimide/cystamine, N-cyanoimidazole,dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) and otherreducing agents as well as external stimuli like ultraviolet light, heatand/or pressure changes.

As is outlined herein, the ligation moieties of the invention may take avariety of configurations, depending on a number of factors. Most of thechemistries depicted herein are used in phosphoramidite reactions thatgenerally progress in a 3′ to 5′ direction. That is, the resin containschemistry allowing attachment of phosphoramidites at the 5′ end of themolecule. However, as is known in the art, phosphoramidites can be usedto progress in the 5′ to 3′ direction; thus, the invention includesmoieties with opposite orientation to those outlined herein.

Each set of ligation probes (or transfer probes) contains a set of afirst ligation moiety and a second ligation moiety. The identificationof these ligation moiety pairs depends on the chemistry of the ligationto be used. In addition, as described herein, linkers (including but notlimited to destabilization linkers) may be present between the probedomain and the ligation moiety of one or both ligation probes. Ingeneral, for ease of discussion, the description herein may use theterms “upstream” and “downstream” ligation probes, although this is notmeant to be limiting.

Different sets of ligation probes can be designed for use in any of theassays and methods described herein. The sets of ligation probes can bedesigned to include ligation probes directed to one or more targetdomains of a target nucleic acid, or different probe sets can bedesigned to detect different target nucleic acids. For example, fordetection of a particular target nucleic acid in embodiments in whichtwo ligation probes are used, a set of ligation probes is designed suchthat the first probe (e.g., the “upstream probe”) comprises a probedomain directed to a first target domain. The set also includes a secondligation probe (e.g., the “downstream probe”) that is directed to asecond target domain that is adjacent to and downstream from the firsttarget domain. The first and second target domains may in someembodiments be separated by a gap of one, two or three nucleotides. Thisset of ligation probes thus produces ligation products that can bedetected to identify the presence of the target nucleic acid to whichthey are directed. In further embodiments, different sets of ligationprobes can be designed to detect different target nucleic acids. Instill further embodiments, different sets of ligation probes aredesigned to detect the same target nucleic acid—in such embodiments, thedifferent sets of ligation probes are directed to different domains ofthe same target nucleic acid, such that the same target nucleic acid mayhave multiple ligation products, depending on the number of differentprobe sets used. As will be appreciated, similar designs can be usedwhen designing sets of probes for use in embodiments in which more thantwo ligation probes are used to form a single ligation product.

Halo Leaving Group Chemistry

In one embodiment of the invention, the chemistry is based on 5′ halogenleaving group technology such as is generally described in Gryanov, S.M., and Letsinger, R. L., (1993) Nucleic Acids Research, 21:1403; Xu, Y.and Kool, E. T. (1997) Tetrahedron Letters, 38:5595; Xu, Y. and Kool, E.T., (1999) Nucleic Acids Research, 27:875; Arar et al., (1995), BioConj.Chem., 6:573; Kool, E. T. et. al, (2001) Nature Biotechnol 19:148; Kool,E. T. et. al., (1995) Nucleic Acids Res, 23 (17):3547; Letsinger et al.,U.S. Pat. No. 5,476,930; Shouten et al., U.S. Pat. No. 6,955,901;Andersen et al., U.S. Pat. No. 7,153,658, all of which are expresslyincorporated by reference herein. In this embodiment, the first ligationprobe includes at its 5′ end a nucleoside having a 5′ leaving group, andthe second ligation probe includes at its 3′ end a nucleoside having 3′nucleophilic group such as a 3′ thiophosphoryl. The 5′ leaving group caninclude many common leaving groups know to those skilled in the artincluding, for example the halo-species (I, Br, Cl) and groups such asthose described by Abe and Kool, J. Am. Chem. Soc. (2004)126:13980-13986, which is incorporated herein by reference in itsentirety. In a more preferred embodiment of this aspect of theinvention, the first ligation probe has a 5′ leaving group attachedthrough a flexible linker and a downstream oligonucleotide which has a3′ thiophosphoryl group. This configuration leads to a significantincrease in the rate of reaction and results in multiple copies ofligated product being produced for every target.

The “upstream” oligonucleotide, defined in relation to the 5′ to 3′direction of the polynucleotide template as the oligonucleotide thatbinds on the “upstream” side (i.e., the left, or 5′ side) of thetemplate includes, as its 5′ end, a 5′-leaving group. Any leaving groupcapable of participating in an S_(N)2 reaction involving sulfur,selenium, or tellurium as the nucleophile can be utilized. The leavinggroup is an atom or group attached to carbon such that on nucleophilicattack of the carbon atom by the nucleophile (sulfur, selenium ortellurium) of the modified phosphoryl group, the leaving group leaves asan anion. Suitable leaving groups include, but are not limited to ahalide, such as iodide, bromide or chloride, a tosylate,benzenesulfonate or p-nitrophenylester, as well as RSO₃ where R isphenyl or phenyl substituted with one to five atoms or groups comprisingF, Cl, Br, I, alkyl (C1 to C6), nitro, cyano, sulfonyl and carbonyl, orR is alkyl with one to six carbons. The leaving group is preferably aniodide, and the nucleoside at the 5′ end of the upstream oligonucleotideis, in the case of DNA, a 5′-deoxy-5′-iodo-2′-deoxynucleoside. Examplesof suitable 5′-deoxy-5′-iodo-2′-deoxynucleosides include, but are notlimited to, 5′-deoxy-5′-iodothymidine (5′-I-T),5′-deoxy-5′-iodo-2′-deoxycytidine (5′-I-dC),5′-deoxy-5′-iodo-2′-deoxyadenosine (5′-I-dA),5′-deoxy-5′-iodo-3-deaza-2′-deoxyadenosine (5′-I-3-deaza-dA),5′-deoxy-5′-iodo-2′-deoxyguanosine (5′-I-dG) and5′-deoxy-5′-iodo-3-deaza-2′-deoxyguanosine (5′-I-3-deaza-dG), and thephosphoroamidite derivatives thereof (see FIG. 2). In the case of RNAoligonucleotides, analogous examples of suitable5′-deoxy-5′-iodonucleosides include, but are not limited to,5′-deoxy-5′-iodouracil (5′-I-U), 5′-deoxy-5′-iodocytidine (5′-I-C),5′-deoxy-5′-iodoadenosine (5′-I-A), 5′-deoxy-5′-iodo-3-deazaadenosine(5′-I-3-deaza-A), 5′-deoxy-5′-iodoguanosine (5′-I-G) and5′-deoxy-5′-iodo-3-deazaguanosine (5′-I-3-deaza-G), and thephosphoroamidite derivatives thereof. In a preferred embodiment, anupstream ligation probe contains 2′-deoxyribonucleotides except that themodified nucleotide on the 5′ end, which comprises the 5′ leaving group,is a ribonucleotide. This embodiment of the upstream nucleotide isadvantageous because the bond between the penultimate2′-deoxyribonucleotide and the terminal 5′ ribonucleotide is susceptibleto cleavage using base. This allows for potential reuse of anoligonucleotide probe that is, for example, bound to a solid support, asdescribed in more detail below. In reference to the CLPA assay, which isdescribed more fully below, the 5′ leaving group of the “upstream” probeis most preferably DABSYL.

The “downstream” oligonucleotide, which binds to the polynucleotidetemplate “downstream” of, i.e., 3′ to, the upstream oligonucleotide,includes, as its 3′ end, a nucleoside having linked to its 3′ hydroxyl aphosphorothioate group (i.e., a “3′-phosphorothioate group”), aphosphoroselenoate group (i.e., a “3′-phosphoroselenoate group), or aphosphorotelluroate group (i.e., a “3′-phosphorotelluroate group”). Thechemistries used for autoligation are thus sulfur-mediated,selenium-mediated, or tellurium mediated. Self-ligation yields aligation product containing a 5′ bridging phosphorothioester(—O—P(O)(O.sup.−)—S—), phosphoroselenoester (—O—P(O)(O.sup.−)—Se—) orphosphorotelluroester (—O—P(O)(O.sup.−)—Te—), as dictated by the groupcomprising the 3′ end of the downstream oligonucleotide. Thisnon-natural, achiral bridging diester is positioned between two adjacentnucleotides and takes the place of a naturally occurring 5′ bridgingphosphodiester. Surprisingly, the selenium-mediated ligation is 3 to 4times faster than the sulfur-mediated ligation, and theselenium-containing ligation product was very stable, despite the lowerbond strength of the Se—P bond. Further, the bridgingphosphoroselenoester, as well as the bridging phosphorotelluroester, areexpected to be cleavable selectively by silver or mercuric ions undervery mild conditions (see Mag et al., Nucleic Acids Res. (1991) 19:14371441).

In one embodiment, a downstream oligonucleotide contains2′-deoxyribonucleotides except that the modified nucleotide on the 3′end, which comprises the 3′ phosphorothioate, phosphoroselenoate, orphosphorotelluroate, is a ribonucleotide. This embodiment of theupstream nucleotide is advantageous because the bond between thepenultimate 2′-deoxyribonucleotide and the terminal ribonucleotide issusceptible to cleavage using base, allowing for potential reuse of anoligonucleotide probe that is, for example, bound to a solid support. Inreference to the CLPA assay, as described more fully below, the“downstream” probe most preferably includes at its 3′ end3′-phosphorothioate.

It should be noted that the “upstream” and “downstream” oligonucleotidescan, optionally, constitute the two ends of a single oligonucleotide, inwhich event ligation yields a circular ligation product. The bindingregions on the 5′ and 3′ ends of the linear precursor oligonucleotidemust be linked by a number of intervening nucleotides sufficient toallow binding of the 5′ and 3′ binding regions to the polynucleotidetarget.

Compositions provided by the invention include a5′-deoxy-5-′iodo-2′-deoxynucleoside, for example a5′-deoxy-5′-iodothymidine (5′-I-T), 5′-deoxy-5′-iodo-2′-deoxycytidine(5′-I-dC), 5′-deoxy-5′-iodo-2′-deoxyadenosine (5′-I-dA),5′-deoxy-5′-iodo-3-deaza-2′-deoxyadenosine (5′-I-3-deaza-dA),5′-deoxy-5′-iodo-2′-deoxyguanosine (5′-I-dG) and5′-deoxy-5′-iodo-3-deaza-2′-deoxyguanosine (5′-I-3-deaza-dG), and thephosphoroamidite derivatives thereof, as well as an oligonucleotidecomprising, as its 5′ end, a 5′-deoxy-5′-iodo-2′-deoxynucleoside of theinvention. Compositions provided by the invention further include a5′-deoxy-5′-iodonucleoside such as 5′-deoxy-5′-iodouracil (5′-I-U),5′-deoxy-5′-iodocytidine (5′-I-C), 5′-deoxy-5′-iodoadenosine (5′-1-A),5′-deoxy-5′-iodo-3-deazaadenosine (5′-I-3-deaza-A),5′-deoxy-5′-iodoguanosine (5′-I-G) and 5′-deoxy-5′-iodo-3-deazaguanosine(5′-I-3-deaza-G), and the phosphoroamidite derivatives thereof, as wellas an oligonucleotide comprising, as its 5′ end, a5′-deoxy-5′-iodonucleoside of the invention. Also included in theinvention is a nucleoside comprising a 3′-phosphoroselenoate group or a3′-phosphorotelluroate group, and an oligonucleotide comprising as its3′ end a nucleoside comprising a 3′-phosphoroselenoate group or a3′-phosphorotelluroate group. Oligonucleotides containing either or bothof these classes of modified nucleosides are also included in theinvention, as are methods of making the various nucleosides andoligonucleotides. Oligonucleotides that are modified at either or bothof the 5′ or 3′ ends in accordance with the invention optionally, butneed not, include a detectable label, preferably a radiolabel, afluorescence energy donor or acceptor group, an excimer label, or anycombination thereof.

In addition, in some cases, substituent groups may also be protectinggroups (sometimes referred to herein as “PG”). Suitable protectinggroups will depend on the atom to be protected and the conditions towhich the moiety will be exposed. A wide variety of protecting groupsare known; for example, DMT is frequently used as a protecting group inphosphoramidite chemistry (as depicted in the figures; however, DMT maybe replaced by other protecting groups in these embodiments. A widevariety of protecting groups are suitable; see for example, Greene'sProtective Groups in Organic Synthesis, herein incorporated by referencefor protecting groups and associated chemistry.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant NH₂, —NHRand —NR₂ groups, with R being as defined herein. In some embodiments,for example in the case of the peptide ligation reactions, primary andsecondary amines find particular use, with primary amines generallyshowing faster reaction rates.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). Aparticular type of sulfur containing moiety is a thioester (—(CO)—S—),usually found as a substituted thioester (—(CO)—SR). By “phosphoruscontaining moieties” herein is meant compounds containing phosphorus,including, but not limited to, phosphines and phosphates. By “siliconcontaining moieties” herein is meant compounds containing silicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCOH groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” herein is meant a —(O—CH₂—CH₂)_(n)— group, althougheach carbon atom of the ethylene group may also be singly or doublysubstituted, i.e. —(O—CR₂—CR₂)_(n)—, with R as described above. Ethyleneglycol derivatives with other heteroatoms in place of oxygen (i.e.—(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or with substitution groups) arealso preferred.

Additionally, in some embodiments, the R group may be a functionalgroup, including quenchers, destabilization moieties and fluorophores(as defined below). Fluorophores of particular use in this embodimentinclude, but are not limited to Fluorescein and its derivatizes, TAMRA(Tetramethyl-6-carboxyrhodamine), Alexa dyes, and Cyanine dyes (e.g. Cy3and Cy5).

Quencher moieties or molecules are known in the art, and are generallyaromatic, multiring compounds that can deactivate the excited state ofanother molecule. Fluorophore-quencher pairs are well known in the art.Suitable quencher moieties include, but are not limited to Dabsyl(Dimethylamini(azobenzene) sulfonyl) Dabcyl(Dimethylamino(azobenzene)carbonyl), Eclipse Quenchers (Glen ResearchCatalog) and blackhole Quenchers (BHQ-1, BHQ-2 and BHQ-3) from BiosearchTechnologies.

Methods of making probes having halo leaving groups is known in the art;see for example Abe et al., Proc Natl Aced Sci USA (2006) 103(2):263-8;Silverman et al., Nucleic Acids Res. (2005) 33(15):4978-86; Cuppollettiet al., Bioconjug Chem. (2005) 16(3):528-34; Sando et al., J Am Chem.Soc. (2004) 4; 126(4):1081-7; Sando et al., Nucleic Acids Res Suppl.(2002) 2:121-2; Sando et al., J Am Chem. Soc. (2002) 124(10):2096-7; Xuet al., Nat. Biotechnol. (2001) 19(2):148-52; Xu et al., Nucleic AcidsRes. (1998) 26(13):3159-64; Moran et al., Proc Natl Aced Sci USA (1997)94(20):10506-11; Kool, U.S. Pat. No. 7,033,753; Kool, U.S. Pat. No.6,670,193; Kool, U.S. Pat. No. 6,479,650; Kool, U.S. Pat. No. 6,218,108;Kool, U.S. Pat. No. 6,140,480; Kool, U.S. Pat. No. 6,077,668; Kool, U.S.Pat. No. 5,808,036; Kool, U.S. Pat. No. 5,714,320; Kool, U.S. Pat. No.5,683,874; Kool, U.S. Pat. No. 5,674,683; and Kool, U.S. Pat. No.5,514,546, each of which is incorporated herein by reference in itsentirety.

Nucleophile Ligation Moieties

In some embodiments, ligation probes of the invention comprise aligation moiety comprising a nucleophile such as an amine. Ligationmoieties comprising both a thiol and an amine find particular use incertain reactions. In general, the nucleophile ligation moieties caninclude a wide variety of potential amino, thiol compounds as long asthe nucleophile ligation moiety contains a thiol group that is proximalto a primary or secondary amino and the relative positioning is suchthat at least a 5 or 6 member ring transition state can be achieveduring the S to N acyl shift.

Accordingly, nucleophile ligation molecules that comprise 1, 2 or 1, 3amine thiol groups find particular use. Primary amines find use in someembodiments when reaction time is important, as the reaction time isgenerally faster for primary than secondary amines, although secondaryamines find use in acyl transferase reactions that contribute todestabilization as discussed below. The carbons between the amino andthiol groups can be substituted with non-hydrogen R groups, althoughgenerally only one non-hydrogen R group per carbon is utilized.Additionally, adjacent R groups may be joined together to form cyclicstructures, including substituted and unsubstituted cycloalkyl and arylgroups, including heterocycloalkyl and heteroaryl and the substitutedand unsubstituted derivatives thereof. In the case where a 1,2 aminothiol group is used and adjacent R groups are attached, it is generallypreferred that the adjacent R groups form cycloalkyl groups (includingheterocycloalkyl and substituted derivatives thereof) rather than arylgroups.

In this embodiment, for the generation of the 4 sigma bond contractionof the chain for destabilization, the replacement ligation moiety relieson an acyl transferase reaction.

Linkers

In many embodiments, linkers (sometimes shown herein as “L” or“-(linker)_(n)-), (where n is zero or one) may optionally be included ata variety of positions within the ligation probe(s). Suitable linkersinclude alkyl and aryl groups, including heteroalkyl and heteroaryl, andsubstituted derivatives of these. In some instances, for example whenNative Peptide Ligation reactions are done, the linkers may be aminoacid based and/or contain amide linkages. As described herein, somelinkers allow the ligation probes to be separated by one or morenucleobases, forming abasic sites within the ligation product, whichserve as destabilization moieties, as described below.

Destabilization Moieties

In accordance with one aspect of the invention, it is desirable toproduce multiple copies of ligated product for each target moleculewithout the aid of an enzyme. One way to achieve this goal involves theligated product disassociating from the target following the chemicalligation reaction to allow a new probe set to bind to the target. Toincrease ligation product turnover, probe designs, instrumentation, andchemical ligation reaction chemistries that increase productdisassociation from the target molecule are desirable. Suitabledestabilization moieties are discussed below and include, but are notlimited to molecule entities that result in a decrease in the overallbinding energy of an oligonucleotide to its target site. Potentialexamples include, but are not limited to alkyl chains, chargedcomplexes, and ring structures.

Previous work has shown one way to achieve product disassociation andincrease product turnover is to “heat cycle” the reaction mixture. Heatcycling is the process of varying the temperature of a reaction so as tofacilitate a desired outcome. Most often heat cycling takes the form ofbriefly raising the temperature of the reaction mixture so that thereaction temperature is above the melting temperature of the ligatedproduct for a brief period of time causing the product to disassociatefrom the target. Upon cooling, a new set of probes is able to bind thetarget, and undergo another ligation reaction. This heat cyclingprocedure has been practiced extensively for enzymatic reactions likePCR.

While heat cycling is one way to achieve product turnover, it ispossible to design probes such that there is significant productturnover without heat cycling. Probe designs and ligation chemistriesthat help to lower the melting temperature of the ligated productincrease product turnover by decreasing product inhibition of thereaction cycle.

Accordingly, in one aspect, the probes are designed to include elements(e.g. destabilization moieties), which, upon ligation of the probes,serve to destabilize the hybridization of the ligation product to thetarget sequence. As a result, the ligated substrate disassociates afterligation, resulting in a turnover of the ligation product, e.g. theligation product comprising the two ligation probes dehybridizes fromthe target sequence, freeing the target sequence for hybridization toanother probe set.

In addition, increasing the concentration of the free (e.g.unhybridized) ligation probes can also help drive the equilibriumtowards release of the ligation product (or transfer product) from thetarget sequence. Accordingly, some embodiments of the invention useconcentrations of probes that are 1,000,000 fold higher than that of thetarget while in other embodiments the probes are 10,000 to 100 foldhigher than that of the target. As will be appreciated by those skilledin the art, increasing the concentration of free probes can be used byitself or with any embodiment outlined herein to achieve productturnover (e.g. amplification). While increasing the probe concentrationcan result in increased product turnover, it can also lead tosignificant off target reactions such as probe hydrolysis and non-targetmediated ligation.

In one aspect, probe elements include structures which lower the meltingtemperature of the ligated product. In some embodiments, probe elementsare designed to hybridize to non-adjacent target nucleobases, e.g. thereis a “gap” between the two hybridized but unligated probes. In general,this is done by using one or two linkers between the probe domain andthe ligation moiety. That is, there may be a linker between the firstprobe domain and the first ligation moiety, one between the second probedomain and the second ligation moiety, or both. In some embodiments, thegap comprises a single nucleobase, although more can also be utilized asdesired. As will be appreciated by those skilled in the art, there maybe a tradeoff between reaction kinetics and length of the linkers; ifthe length of the linker(s) are so long that contact resulting inligation is kinetically disfavored, shorter linkers may be desired.However, in some cases, when kinetics are not important, the length ofthe gap and the resulting linkers may be longer, to allow spanning gapsof 1 to 10 nucleobases. Generally, in this embodiment, what is importantis that the length of the linker(s) roughly corresponds to the number ofnucleobases in the gap.

In another aspect of this embodiment of the invention, the formation ofabasic sites in a ligation product as compared to the target sequenceserves to destabilize the duplex. For example, Abe and Kool (J. Am.Chem. Soc. (2004) 126:13980-13986) compared the turnover when twodifferent 8-mer oligonucleotide probes (Bu42 and DT40) were ligated withthe same 7-mer probe (Thio 4). When Thio4 is ligated with DT40, acontinuous 15-mer oligonucleotide probe with a nearly native DNAstructure is formed that should be perfectly matched with the DNAtarget. However, when Thio4 is ligated with Bu42, a 15-meroligonucleotide probe is formed, but when the probe is bound to thetarget, it has an abasic site in the middle that is spanned by an alkanelinker. Comparison of the melting temperature (Tm) of these two probeswhen bound to the target shows approximately a 12° C. difference inmelting temperature (58.5 for Bu42 versus 70.7° C. for DT40). This 12°C. difference in melting temperature led to roughly a 10-fold increasein product turnover (91.6-Bu42 versus 8.2 DT40) at 25° C. when the probesets (10.000-fold excess, 10 μM conc) were present in large excesscompared to the target (1 nM). Similarly, Dose et al (Dose 2006) showedhow a 4° C. decrease in Tm for two identical sequences, chemicallyligated PNA probes (53° C. versus 57° C.) results in approximately a4-fold increase in product turnover.

Recent work has demonstrated the use of chemical ligation based QuenchedAuto-Ligation (QUAL) probes to monitor RNA expression and detect singlebase mismatches inside bacterial and human cells (WO 2004/0101011 hereinincorporated by reference).

In one embodiment, destabilization moieties are based on the removal ofstabilization moieties. That is, if a ligation probe contains a moietythat stabilizes its hybridization to the target, upon ligation andrelease of the stabilization moiety, there is a drop in the stability ofthe ligation product. Accordingly, one general scheme for reducingproduct inhibition is to develop probes that release a molecular entitylike a minor groove binding molecule during the course of the initialchemical ligation reaction or following a secondary reaction postligation. Depending on the oligonucleotide sequence, minor groovebinders like the dihydropyrroloindole tripeptide (DPI₃) described byKutyavin (Kutyavin 1997 and Kutyavin 2000) can increase the Tm of aduplex nucleic acid by up to 40° C. when conjugated to the end of anoligonucleotide probe. In contrast, the unattached version of the DPI3only increases the Tm of the same duplex by 2° C. or so. Thus, minorgroove binders can be used to produce probe sets with enhanced bindingstrengths, however if the minor groove binder is released during thecourse of the reaction, the binding enhancement is loss and the ligatedproduct will display a decreased Tm relative to probes in which theminor groove binder is still attached.

Suitable minor groove binding molecules include, but are not limited to,dihydropyrroloindole tripeptide (DPI₃), distamycin A, andpyrrole-imidazole polyamides (Gottesfeld, J. M., et al., J. Mol. Biol.(2001) 309:615-629.

In addition to minor groove binding molecules tethered intercalators andrelated molecules can also significantly increase the meltingtemperature of oligonucleotide duplexes, and this stabilization issignificantly less in the untethered state. (Dogan, et al., J. Am. Chem.Soc. (2004) 126:4762-4763 and Narayanan, et al., Nucleic Acids Research,(2004) 32:2901-2911).

Similarly, as will be appreciated by those in the art, probes withattached oligonucleotide fragments (DNA, PNA, LNA, etc) capable oftriple helix formation, can serve as stabilization moieties that uponrelease, results in a decrease of stabilization of the ligation productto the target sequence (Pooga, M, et al., Biomolecular Engineering(2001) 17:183-192.

Another general scheme for decreasing product inhibition by lowering thebinding strength of the ligated product is to incorporate abasic sitesat the point of ligation. This approach has been previously demonstratedby Abe (J. Am. Chem. Soc. (2004) 126:13980-13986), however it is alsopossible to design secondary probe rearrangements to further amplify thedecrease in Tm via straining the alignment between the ligated probesand the target. For example, Dose et al. (Org. Letters (2005) 7:204365-4368) showed how a rearrangement post-ligation that changed thespacing between PNA bases from the ideal 12 sigma bonds to 13 resultedin a lowering of the Tm by 4° C. Larger rearrangements and secondaryreactions that interfere with the binding of the product to the targetor result in the loss of oligonucleotide bases can further decrease theTm.

The present invention provides methods and compositions for a ligationreaction that results in a chain contraction of up to 4 sigma bondsduring the rearrangement, which should have a significant effect on theTm post-rearrangement compared to the 1 base expansion using thechemistry described by Dose. This chemistry is based on the acyltransfer auxiliary that has been described previously (Offer et al., JAm Chem. Soc. (2002) 124(17):4642-6). Following completion of the chaincontraction, a free-thiol is generated that is capable of undergoinganother reaction either with a separate molecule or with itself. Forexample, this thiol could react with an internal thioester to severelykink the oligonucleotide and thus further decrease the ligationproduct's ability to bind to the target.

Thus, in this embodiment, ligation reactions that release functionalgroups that will undergo a second reaction with the ligation product canreduce stabilization of the hybrid of the ligation product and thetarget sequence.

Variable Spacer Sequences

In addition to the target domains, ligation moieties, and optionallinkers, one or more of the ligation probes of the invention can havevariable spacer sequences (also referred to as “stuffer sequences”).These variable spacer sequences are of particular use in the CLPA-CEassays described in further detail herein. That is, the variable spacersequence can serve as a type of “label” or “barcode” to identify thetarget sequence when the products are detected on the basis of length,for example using capillary electrophoresis (CE).

Variable spacer sequences are domains of ligation probes that can havevarying lengths. These varying lengths will in some embodiments bespecific to a particular target sequence to which other domains of theligation probe (e.g., the probe domain) are able to hybridize, such thatthe length of the ligation product resulting from ligation of ligationprobes containing stuffer sequences is a target-specific length. As aresult, any amplicons generated from such ligation products will alsohave a target-specific length. In other embodiments, the variable spacersequence is designed to render all ligation products of similar lengthso as to facilitate the efficiency of subsequent amplificationreactions.

In some embodiments, only one of the ligation probes of a ligation probepair comprises a variable spacer sequence. In other embodiments, bothligation probes comprise the variable spacer sequence. In embodiments inwhich three or more ligation probes are used, one or more of theligation probes forming a particular ligation product can contain thevariable spacer sequence, although in this embodiment is it generallyone or both of the terminal probes that contains the variable spacersequence.

In further embodiments and in accordance with any of the above, thevariable spacer sequence is contained within a region of the ligationprobe that is substantially non-complementary to both any other domainsof the target nucleic acid as well as to any of the target nucleic acidsin a sample.

In still further embodiments, the ligation probes (as will be discussedin further detail below) contain a primer sequence. In accordance withany of the above, the variable spacer sequence is in some embodimentscontained between the probe domain (the region of the ligation probethat hybridizes to a target sequence) and the primer sequence, such thatthe amplicon contains the variable spacer sequence.

In further aspects, variable spacer sequences of the invention includenucleic acids such as DNA or RNA. Variable spacer sequences may alsoinclude a combination of both deoxyribonucleotides and ribonucleotides.Variable spacer sequences may further include nucleotide analogues,linkers, or any of the additional moieties discussed herein asoptionally present in ligation and/or target capture probes of theinvention.

In general, for multiplex assays where multiple target sequences are tobe detected simultaneously, variable spacer sequences are designed toresult in either or both of the ligation product or (in the case wherean amplification reaction follows the ligation step) the resultingamplicons from each ligation product to have a different nucleotidelength from other ligation products or amplicons in the sample. That is,the amplicon from one target nucleic acid will be different from thelength of the amplicon from a different target nucleic acid. Similarly,when multiple ligation probe sets are used for a single target as isshown in FIGS. 6 and 11, each ligation product and/or amplicon will havea different length, allowing detection of the different products.

The differing lengths of the variable spacer sequences will depend onthe sensitivity of the detection system. For sophisticated Capillaryelectrophoresis (CE) systems, such as Genetic Analyzer systems made byLife Technologies or the PACE systems by Beckman Coulter, ligationproducts and/or amplicons can be separated and thus detected when theydiffer in length by as few as one nucleotide. Other CE systems withlower size resolution, may require length differences of 5 to 100 basepairs depending on the resolution. In general, higher resolution CEsystems require longer separation channel lengths and often longerseparation times. Furthermore, denaturing capillary electrophoresissystems tend to have better resolution than non-denaturing systems. Ingeneral, the variable spacer sequences are designed to result innucleotide differences ranging from 1 to 100 bases with 5 to 20 basesbeing preferred. Greater size differences can be used, but often requireadditional cost or reduce the number of possible ligation probes thatcan be combined in a single test.

In addition, variable spacer sequences can be used in conjunction withother labels to expand the number of different “barcodes” that can beused. That is, a variable spacer sequence length can be “reused” byencoding it with a second label; for example, one amplicon containing avariable spacer sequence of 20 nucleotides can use a fluorescent labelof a first color, and another amplicon containing the 20 mer spacersequence can use a fluorescent label of a different color. Theseamplicons of the same size can be simultaneously detected using amulti-channel CE instrument that can identify amplicons with differentwavelength (color) products like is commonly practiced in forensicmedicine.

Additional Functionalities of Ligation Probes

In addition to the target domains, ligation moieties, and optionallinkers, one or more of the ligation probes of the invention can haveadditional functionalities, including, but not limited to, promoter andprimer sequences (or complements thereof, depending on the assay),labels, including label probe binding sequences, and capture or anchorsequences.

In many embodiments, the ligation probes are constructed so as tocontain the necessary priming site or sites for the subsequentamplification scheme. In a preferred embodiment the priming sites areuniversal priming sites. By “universal priming site” or “universalpriming sequences” herein is meant a sequence of the probe that willbind a primer for amplification.

In a preferred embodiment, one universal priming sequence or site isused. In this embodiment, a preferred universal priming sequence is theRNA polymerase T7 sequence, that allows the T7 RNA polymerase make RNAcopies of the adapter sequence as outlined below. Additional disclosureregarding the use of T7 RNA polymerase is found in U.S. Pat. Nos.6,291,170, 5,891,636, 5,716,785, 5,545,522, 5,922,553, 6,225,060 and5,514,545, all of which are expressly incorporated herein by reference.

In a preferred embodiment, for example when amplification methodsrequiring two primers such as PCR are used, each probe preferablycomprises an upstream universal priming site (UUP) and a downstreamuniversal priming site (DUP). Again, “upstream” and “downstream” are notmeant to convey a particular 5′-3′ orientation, and will depend on theorientation of the system. Preferably, only a single UUP sequence and asingle DUP sequence is used in a probe set, although as will beappreciated by those in the art, different assays or differentmultiplexing analysis may utilize a plurality of universal primingsequences. In some embodiments probe sets may comprise differentuniversal priming sequences. In addition, the universal priming sitesare preferably located at the 5′ and 3′ termini of the target probe (orthe ligated probe), as only sequences flanked by priming sequences willbe amplified.

In addition, universal priming sequences are generally chosen to be asunique as possible given the particular assays and host genomes toensure specificity of the assay. However, as will be appreciated bythose in the art, sets of priming sequences/primers may be used; thatis, one reaction may utilize 500 target probes with a first primingsequence or set of sequences, and an additional 500 probes with a secondsequence or set of sequences.

As will be appreciated by those in the art, when two priming sequencesare used, the orientation of the two priming sites is generallydifferent. That is, one PCR primer will directly hybridize to the firstpriming site, while the other PCR primer will hybridize to thecomplement of the second priming site. Stated differently, the firstpriming site is in sense orientation, and the second priming site is inantisense orientation.

As will be appreciated by those in the art, in general, highlymultiplexed reactions can be performed, with all of the universalpriming sites being the same for all reactions. Alternatively, “sets” ofuniversal priming sites and corresponding probes can be used, eithersimultaneously or sequentially. The universal priming sites are used toamplify the modified probes to form a plurality of amplicons that arethen detected in a variety of ways, as outlined herein. In preferredembodiments, one of the universal priming sites is a T7 site. In someembodiments this priming site serves as a template for the synthesis ofRNA.

In a preferred embodiment, when detecting multiple targetssimultaneously, all of the oligonucleotide ligation probe sets in thereaction are designed to contain identical promoter or primer pairbinding sites such that following ligation and purification, ifappropriate, all of the ligated products can be amplified simultaneouslyusing the same enzyme and/or same primers. In other words, inembodiments involving the detection of multiple target nucleic acids,different ligation probe sets containing ligation probes directed todifferent target nucleic acid sequences can in some embodiments possessidentical promoter or primer pair binding sites (e.g., “universal”primer binding sites) such that the ligation products resulting from thehybridization and ligation of these ligation probes can be amplifiedusing the same enzyme and/or primers.

In one embodiment, one or more of the ligation probes comprise apromoter sequence. In embodiments that employ a promoter sequence, thepromoter sequence or its complement will be of sufficient length topermit an appropriate polymerase to interact with it. Detaileddescriptions of sequences that are sufficiently long for polymeraseinteraction can be found in, among other places, Sambrook and Russell,which are hereby incorporated by reference for all purposes and inparticular for all teachings related to promoter sequences forpolymerases. In certain embodiments, amplification methods comprise atleast one cycle of amplification, for example, but not limited to, thesequential procedures of: interaction of a polymerase with a promoter;synthesizing a strand of nucleotides in a template-dependent mannerusing a polymerase; and denaturing the newly-formed nucleic acid duplexto separate the strands.

In another embodiment, one or both of the ligation probes comprise aprimer sequence. As outlined below, the ligation products of the presentinvention may be used in additional reactions such as enzymaticamplification reactions. In one embodiment, the ligation probes includeprimer sequences designed to allow an additional level of amplification.As used herein, the term “primer” refers to nucleotide sequence, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofnucleic acid sequence synthesis when placed under conditions in whichsynthesis of a primer extension product which is complementary to anucleic acid strand is induced, i.e. in the presence of differentnucleotide triphosphates and a polymerase in an appropriate buffer(“buffer” includes pH, ionic strength, cofactors etc.) and at a suitabletemperature. One or more of the nucleotides of the primer can bemodified, for instance by addition of a methyl group, a biotin ordigoxigenin moiety, a fluorescent tag or by using radioactivenucleotides. A primer sequence need not reflect the exact sequence ofthe template. For example, a non-complementary nucleotide fragment maybe attached to the 5′ end of the primer, with the remainder of theprimer sequence being substantially complementary to the target strand.

By using several priming sequences and primers, a first ligation productcan serve as the template for additional ligation products. These primersequences may serve as priming sites for PCR reactions, which can beused to amplify the ligation products. In addition to PCR reactions,other methods of amplification can utilize the priming sequences,including but not limited to ligase chain reactions, Invader™,positional amplification by nick translation (NICK), primerextension/nick translation, and other methods known in the art. As usedherein, “amplification” refers to an increase in the number of copies ofa particular nucleic acid. Copies of a particular nucleic acid made invitro in an amplification reaction are called “amplicons” or“amplification products”.

Amplification may also occur through a second ligation reaction, inwhich the primer sites serve as hybridization sites for a new set ofligation probes which may or may not comprise sequences that areidentical to the first set of ligation probes that produced the originalligation products. The target sequence is thus exponentially amplifiedthrough amplification of ligation products in subsequent cycles ofamplification.

In another embodiment of this aspect of the invention, the primersequences are used for nested ligation reactions. In such nestedligation reactions, a first ligation reaction is accomplished usingmethods described herein such that the ligation product can be captured,for example by using biotinylated primers to the desired strand andcapture on beads (particularly magnetic beads) coated with streptavidin.After the ligation products are captured, a second ligation reaction isaccomplished by hybridization of ligation probes to primer sequenceswithin a section of the ligation product which is spatially removed from(i.e., downstream from) the end of the ligation product which isattached to the capture bead, probe, etc. At least one of the primersequences for the secondary ligation reaction will be located within theregion of the ligation product complementary to the ligation probe whichis not the ligation probe that included the anchor or capture sequence.The ligation products from this second ligation reaction will thusnecessarily only result from those sequences successfully formed fromthe first chemical ligation, thus removing any “false positives” fromthe amplification reaction. In another embodiment, the primer sequencesused in the secondary reaction may be primer sites for other types ofamplification reactions, such as PCR.

In one embodiment, one or more of the ligation probes comprise an anchorsequence. By “anchor sequence” herein is meant a component of a ligationprobe that allows the attachment of a ligation product to a support forthe purposes of detection. Suitable means for detection include asupport having attached thereto an appropriate capture moiety.Generally, such an attachment will occur via hybridization of the anchorsequence with a capture probe, which is substantially complementary tothe anchor sequence.

In a preferred embodiment, one of the probes further comprises an“anchor” sequence, (sometimes referred to in the art and herein as “zipcodes” or “bar codes” or “adapters”). Adapters facilitate immobilizationof probes to allow the use of “universal arrays”. That is, arrays(either solid phase or liquid phase arrays) are generated that containcapture probes that are not target specific, but rather specific toindividual (preferably) artificial adapter sequences.

Thus, an “adapter sequence” is a nucleic acid that is generally notnative to the target sequence, i.e. is exogenous, but is added orattached to the target sequence. It should be noted that in thiscontext, the “target sequence” can include the primary sample targetsequence, or can be a derivative target such as a reactant or product ofthe reactions outlined herein; thus for example, the target sequence canbe a PCR product, a first ligation probe or a ligated probe in an OLAreaction, etc. The terms “barcodes”, “adapters”, “addresses”, tags” and“zipcodes” have all been used to describe artificial sequences that areadded to amplicons to allow separation of nucleic acid fragment pools.One preferred form of adapters are hybridization adapters. In thisembodiment adapters are chosen so as to allow hybridization to thecomplementary capture probes on a surface of an array. Adapters serve asunique identifiers of the probe and thus of the target sequence. Ingeneral, sets of adapters and the corresponding capture probes on arraysare developed to minimize cross-hybridization with both each other andother components of the reaction mixtures, including the targetsequences and sequences on the larger nucleic acid sequences outside ofthe target sequences (e.g. to sequences within genomic DNA). Other formsof adapters are mass tags that can be separated using mass spectroscopy,electrophoretic tags that can be separated based on electrophoreticmobility, etc. Some adapter sequences are outlined in U.S. Ser. No.09/940,185, filed Aug. 27, 2001, hereby incorporated by reference in itsentirety. Preferred adapters are those that meet the following criteria.They are not found in a genome, preferably a human genome, and they donot have undesirable structures, such as hairpin loops.

In one embodiment the use of adapter sequences allow the creation ofmore “universal” surfaces; that is, one standard array, comprising afinite set of capture probes can be made and used in any application.The end-user can customize the array by designing different solubletarget probes, which, as will be appreciated by those in the art, isgenerally simpler and less costly. In a preferred embodiment, an arrayof different and usually artificial capture probes are made; that is,the capture probes do not have complementarity to known targetsequences. The anchor sequences can then be incorporated in the targetprobes.

As will be appreciated by those in the art, the length of the anchorsequences will vary, depending on the desired “strength” of binding andthe number of different anchor desired. In a preferred embodiment,adapter sequences range from about 6 to about 500 basepairs in length,with from about 8 to about 100 being preferred, and from about 10 toabout 25 being particularly preferred.

In a preferred embodiment, the adapter sequence uniquely identifies thetarget analyte to which the target probe binds. That is, while theadapter sequence need not bind itself to the target analyte, the systemallows for identification of the target analyte by detecting thepresence of the adapter. Accordingly, following a binding orhybridization assay and washing, the probes including the adapters areamplified. Detection of the adapter then serves as an indication of thepresence of the target nucleic acid.

In one embodiment the adapter includes both an identifier region and aregion that is complementary to capture probes on a universal array asdescribed above. In this embodiment, the amplicon hybridizes to captureprobes on a universal array. Detection of the adapter is accomplishedfollowing hybridization with a probe that is complementary to theadapter sequence. Preferably the probe is labeled as described herein.

In general, similar to variable spacer sequences, unique adaptersequences are used for each unique target analyte. That is, theelucidation or detection of a particular adapter sequence allows theidentification of the target analyte to which the target probecontaining that adapter sequence bound. However, as discussed herein, insome cases, it is possible to “reuse” adapter sequences and have morethan one target analyte share an adapter sequence.

In a preferred embodiment the adapters contain different sequences orproperties that are indicative of a particular target molecule. That is,each adapter uniquely identifies a target sequence. As described above,the adapters are amplified to form amplicons. The adapter is detected asan indication of the presence of the target analyte, i.e. the particulartarget nucleic acid.

In one embodiment of this aspect of the invention, the upstreamoligonucleotide is designed to have an additional nucleotide segmentthat does not bind to the target of interest, but is to be used tosubsequently capture the ligated product on a suitable solid support ordevice of some sort. In a preferred embodiment of this aspect of theinvention, the downstream oligonucleotide has a detectable labelattached to it, such that following ligation, the resulting product willcontain a capture sequence for a solid support at its 3′ end and adetectable label at its 5′ end, and only ligated products will containboth the capture sequence and the label.

In another aspect of the invention pertaining in particular to multiplextarget detection, each upstream probe of a probe set may be designed tohave a unique sequence (also referred to herein as an “anchor sequence)at is 3′ end that corresponds to a different position on a DNA array.Each downstream probe of a probe set may optionally contain a detectablelabel that is identical to the other downstream probes, but a uniquetarget binding sequence that corresponds to its respective targets.Following hybridization with the DNA array, only ligated probes thathave both an address sequence (upstream probe) and a label (downstreamprobe) will be observable. In another aspect of the invention, thedetectable label can be attached to the upstream probe and the capturesequence can be a part of the downstream probe, such that the ligatedproducts will have the detectable label more towards the 3′ end and thecapture sequence towards the 5′ end of the ligated product. The exactconfiguration is best determined via consideration of the ease ofsynthesis as well as the characteristics of the devices to be used tosubsequently detect the ligated reaction product.

The anchor sequence may have both nucleic and non-nucleic acid portions.Thus, for example, flexible linkers such as alkyl groups, includingpolyethylene glycol linkers, may be used to provide space between thenucleic acid portion of the anchor sequence and the support surface.This may be particularly useful when the ligation products are large.

In addition, in some cases, sets of anchor sequences that correspond tothe capture probes of “universal arrays” can be used. As is known in theart, arrays can be made with synthetic generic sequences as captureprobes, that are designed to non-complementary to the target sequencesof the sample being analyzed but to complementary to the array bindingsequences of the ligation probe sets. These “universal arrays” can beused for multiple types of samples and diagnostics tests because samearray binding sequences of the probes can be reused/paired withdifferent target recognition sequences.

In one embodiment, one or more of the ligation probes comprise a label.By “label” or “labeled” herein is meant that a compound has at least oneelement, isotope or chemical compound attached to enable the detectionof the compound, e.g. renders a ligation probe or ligation or transferproduct detectable using known detection methods, e.g., electronic,spectroscopic, photochemical, or electrochemiluminescent methods. Ingeneral, labels fall into three classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) magnetic, electrical, thermal; andc) colored or luminescent dyes; although labels include enzymes andparticles such as magnetic particles as well. The dyes may bechromophores or phosphors but are preferably fluorescent dyes, which dueto their strong signals provide a good signal-to-noise ratio. Suitabledyes for use in the invention include, but are not limited to,fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, fluorescein isothiocyanate, carboxyfluorescein(FAM), dichlorotriazinylamine fluorescein, rhodamine,tetramethylrhodamine, umbelliferone, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, LuciferYellow, Cascade Blue™, Texas Red, alexa dyes, dansyl chloride,phycoerythrin, green fluorescent protein and its wavelength shiftedvariants, bodipy, and others known in the art such as those described inHaugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; TheSynthegen catalog (Houston, Tex.), Lakowicz, Principles of FluorescenceSpectroscopy, 2nd Ed., Plenum Press New York (1999), and othersdescribed in the 6th Edition of the Molecular Probes Handbook by RichardP. Haugland, herein expressly incorporated by reference. Additionallabels include nanocrystals or Q-dots as described in U.S. Ser. No.09/315,584, herein expressly incorporated by reference.

In a preferred embodiment, the label is a secondary label that part of abinding partner pair. For example, the label may be a hapten or antigen,which will bind its binding partner. In a preferred embodiment, thebinding partner can be attached to a solid support to allow separationof extended and non-extended primers. For example, suitable bindingpartner pairs include, but are not limited to: antigens (such asproteins (including peptides)) and antibodies (including fragmentsthereof (FAbs, etc.)); proteins and small molecules, includingbiotin/streptavidin; enzymes and substrates or inhibitors; otherprotein-protein interacting pairs; receptor-ligands; and carbohydratesand their binding partners. Nucleic acid-nucleic acid binding proteinpairs are also useful. In general, the smaller of the pair is attachedto the NTP for incorporation into the primer. Preferred binding partnerpairs include, but are not limited to, biotin (or imino-biotin) andstreptavidin, digeoxinin and Abs, and Prolinx™ reagents.

In a preferred embodiment, the binding partner pair comprises biotin orimino-biotin and streptavidin. Imino-biotin is particularly preferred asimino-biotin disassociates from streptavidin in pH 4.0 buffer whilebiotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or90% formamide at 95° C.).

In a preferred embodiment, the binding partner pair comprises a primarydetection label (for example, attached to a ligation probe) and anantibody that will specifically bind to the primary detection label. By“specifically bind” herein is meant that the partners bind withspecificity sufficient to differentiate between the pair and othercomponents or contaminants of the system. The binding should besufficient to remain bound under the conditions of the assay, includingwash steps to remove non-specific binding. In some embodiments, thedissociation constants of the pair will be less than about 10⁻⁴ to 10⁻⁶M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred and 10⁻⁹ M⁻¹being particularly preferred.

In a preferred embodiment, the secondary label is a chemicallymodifiable moiety. In this embodiment, labels comprising reactivefunctional groups are incorporated into the nucleic acid. The functionalgroup can then be subsequently labeled with a primary label. Suitablefunctional groups include, but are not limited to, amino groups, carboxygroups, maleimide groups, oxo groups and thiol groups, with amino groupsand thiol groups being particularly preferred. For example, primarylabels containing amino groups can be attached to secondary labelscomprising amino groups, for example using linkers as are known in theart; for example, homo- or hetero-bifunctional linkers as are well known(see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155 200, incorporated herein by reference).

In this embodiment, the label may also be a label probe binding sequenceor complement thereof. By “label probe” herein is meant a nucleic acidthat is substantially complementary to the binding sequence and islabeled, generally directly.

The compositions of the invention are generally made using knownsynthetic techniques. In general, methodologies based on standardphosphoramidite chemistries find particular use in one aspect of thepresent invention, although as is appreciated by those skilled in theart, a wide variety of nucleic acid synthetic reactions are known. Thespacing of the addition of fluorophores and quenchers is well known aswell.

VI. Target Capture Probes

In certain aspects, and in accordance with any of the description ofligation probes discussed herein, the present invention further includesthe use of target capture probes. These target capture probes do notgenerally participate in the chemical ligation reactions of the ligationprobes, and are useful for increasing the specificity of assays. As isdescribed further herein, the target capture probes are hybridized tothe target nucleic acid either upstream or downstream from the ligationproduct formed from ligation probes as described herein. Hybridizing thetarget capture probe to the target nucleic acid forms a target complexthat comprises the target nucleic acid, one or more ligation products,and the target capture probe. The target capture probe contains acapture moiety that can be used to capture the target complex on a solidsupport or a bead. Following capture on the support or bead, thenon-bound species are removed. The target complex (also referred toherein as a “capture complex”) and/or the ligation products is theneither analyzed or subjected to amplification (such as PCR).

In general, target capture probes of the invention contain a domain thathybridizes to the target nucleic acid and an additional capture moietythat can be used to selectively capture complexes/molecules that arebound to the target capture through hybridization or other interactions.In some embodiments, the capture moiety can be a capture nucleic acidsequence, a bead, or a binding partner of a binding partner pair (suchas biotin, which can then be captured with its binding partnerstreptavidin).

Target capture probes are designed to hybridize upstream or downstreamfrom ligation probes hybridized to target domains of a nucleic acid. Incertain embodiments, target capture probes will hybridize within apredetermined distance to the ligation probes.

As is discussed herein, methods of the invention include thehybridization of two or more ligation probes to target domains of atarget nucleic acid under conditions such that the ligation probesspontaneously undergo a chemical ligation without the addition of aligase enzyme. In embodiments of the invention that use target captureprobes along with ligation probes, it will be appreciated that thetarget capture probes can be hybridized to the target nucleic acid priorto, subsequent to or simultaneously with hybridization of the one ormore ligation probes. Similarly, hybridization of the target captureprobes may also occur prior to, subsequent to or simultaneously with thespontaneous ligation of the ligation probes.

In certain embodiments, multiple target capture probes are applied to atarget nucleic acid. As will be appreciated, these multiple captureprobes can be designed to hybridize to any point along the length of thetarget. In embodiments in which a single ligation product is formed on atarget nucleic acid, multiple ligation probes may be used to flank theligation product on either side. This embodiment is of particular use inquality control or assessments of nucleic acid integrity, because thereis an increased likelihood that even a degraded target nucleic acid willbe captured when multiple target capture probes are used.

In embodiments in which multiple ligation products are formed on atarget nucleic acid, multiple target capture probes may also behybridized to the target nucleic acid. As discussed above, thesemultiple target capture probes may be designed to hybridize at differentpoints along the length of the target nucleic acid. In some embodiments,target capture probes are designed to hybridize at or near the 3′, 5′ orboth the 3′ and 5′ end of the target nucleic acid. In furtherembodiments, the target capture probes are designed to be hybridized toregions upstream, downstream, and/or interleaved between the differentligation products formed on a target nucleic acid.

VII. Assays and Detection Methods

In exemplary aspects, the present invention provides methods fordetecting a plurality of different target nucleic acids in a sample.Such target nucleic acids include at least a first and a second targetdomain, where the first and second target domains are adjacent to eachother. The target nucleic acids can also include a third domain that isa target capture domain, and that target capture domain is locatedupstream or downstream (i.e., 5′ or 3′) to the first and second targetdomains.

In preferred embodiments, assays of the invention include the steps ofproviding ligation substrates that include a target nucleic acidcomprising the target domains and target capture domains discussedabove. These ligation substrates each further include a first set ofligation probes comprising a first and second nucleic acid ligationprobe. The first nucleic acid ligation probe is hybridized to the firsttarget domain of the target nucleic acid sequence, and the secondnucleic acid ligation probe is hybridized to the second target domain ofthe target nucleic acid sequence. In such embodiments, the first targetdomain is upstream of the second target domain. The first nucleic acidligation probe further includes a 5′ ligation moiety and the secondnucleic acid ligation probe further includes a 3′ ligation moiety. Sincethe first and second target domains are adjacent to each other, theligation moieties of the two ligation probes hybridized to the targetdomains are adjacent to each other and are able to undergo ligationwithout the use of a ligase enzyme to form a ligation product.

Embodiments of the invention further include hybridizing a targetcapture probe to the third target capture domain of the target nucleicacid to form a target complex. The target complex thus comprises thetarget nucleic acid, the first ligation probe hybridized to the firsttarget domain, the second ligation probe hybridized to the secondligation domain and the target capture probe hybridized to the thirdtarget capture domain. The target capture probe comprises a capturemoiety, enabling capture of the target complex on a surface or substrate(such as a bead). Capturing the target complex can be used to separatethe target complex from unbound target nucleic acids and ligationprobes—in other words, use of a target capture probes provides a way toisolate those target nucleic acids on which a ligation product hassuccessfully been formed.

Assays of the invention can further include amplifying the ligationproduct formed from the different ligation substrates to form ampliconsand then detecting the amplicons to detect the target nucleic acids.

As will be appreciated, assays of the invention may utilize multiplesets of ligation probes directed to multiple target domains. Forexample, the nucleic acids can further include a fourth target domainadjacent to a fifth target domain, and the ligation substrates furtherinclude a second set of ligation probes that include a third ligationprobe hybridized to the fourth target domain and a fourth ligation probehybridized to the fifth target domain. As with the first and secondligation probes discussed above, the third and fourth ligation probesligate without the use of a ligase enzyme, and thus the target nucleicacid comprises multiple ligation products. Target capture probes canagain be hybridized to the target nucleic acids to form target complexescomprising the multiple ligation products, the target nucleic acid andthe target capture probes. As discussed above, these target complexescan be captured on a surface and the multiple ligation products can insome embodiments be amplified to form amplicons, which can then bedetected.

Similarly, the target nucleic acids can further include a sixth targetdomain adjacent to a seventh target domain, and a third set of ligationprobes containing fifth and sixth ligation probes can be hybridized tothe sixth and seventh target domains respectively. As with any of theligation probes discussed herein, the fifth and sixth ligation probesare ligated without the use of a ligase enzyme, thus producing a targetnucleic acid comprising multiple ligation products (i.e., the ligationproduct formed from the ligation of the first and second ligation probesand/or the ligation product formed from the third and fourth ligationprobes). As will be appreciated, a target nucleic acid may contain anynumber of target domains, and thus any number of ligation probe sets canbe used to form ligation products on that target nucleic acid inaccordance with the present invention.

Any of the above target nucleic acids comprising multiple ligationproducts may further be hybridized with one or more target captureprobes to form target complexes. Those target complexes can then becaptured on a substrate. The ligation products of the target complexesare then amplified using methods known in the art and discussed hereinto form amplicons, and those amplicons are then detected to identifyand/or quantify the presence of target nucleic acids.

In certain embodiments, the ligation products formed in accordance withany of the methods described herein are not amplified but are insteaddirectly detected using any of the methods described herein.

Prior to detecting the ligation or transfer reaction product, there maybe additional amplification reactions. Secondary amplification reactionscan be used to increase the signal for detection of the target sequence;e.g. by increasing the number of ligated products produced per copy oftarget. In one embodiment, any number of standard amplificationreactions can be performed on the ligation product, including, but notlimited to, strand displacement amplification (SDA), nucleic acidsequence based amplification (NASBA), ligation amplification and thepolymerase chain reaction (PCR); including a number of variations ofPCR, including “quantitative competitive PCR” or “QC-PCR”, “arbitrarilyprimed PCR” or “AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strandconformational polymorphism” or “PCR-SSCP”, “reverse transcriptase PCR”or “RT-PCR”, “biotin capture PCR”, “vectorette PCR”. “panhandle PCR”,and “PCR select cDNA subtraction”, among others. In one embodiment, theamplification technique is not PCR. According to certain embodiments,one may use ligation techniques such as gap-filling ligation, including,without limitation, gap-filling OLA and LCR, bridging oligonucleotideligation, FEN-LCR, and correction ligation. Descriptions of thesetechniques can be found, among other places, in U.S. Pat. No. 5,185,243,published European Patent Applications EP 320308 and EP 439182,published PCT Patent Application WO 90/01069, published PCT PatentApplication WO 02/02823, and U.S. patent application Ser. No.09/898,323.

In addition to standard enzymatic amplification reactions, it ispossible to design probe schemes where the ligated product that isinitially produced can itself be the target of a secondary chemicalligation reaction.

Furthermore, “preamplification reactions” can be done on starting samplenucleic acids to generate more target sequences for the chemicalreaction ligation. For example, whole genome amplification can be done.

As will be appreciated by those skilled in the art, assays utilizingmethods and compositions of the invention can take on a wide variety ofconfigurations, depending on the desired application, and can include insitu assays (similar to FISH), solution based assays (e.g.transfer/removal of fluorophores and/or quenchers), and heterogeneousassays (e.g. utilizing solid supports for manipulation, removal and/ordetection, such as the use of high density arrays). In addition, assayscan include additional reactions, such as pre-amplification of targetsequences and secondary amplification reactions after ligation hasoccurred, as is outlined herein.

Assays pertaining to this aspect of the invention, as described herein,may rely on increases in a signal, e.g. the generation of fluorescenceor chemiluminescence. However, as will be appreciated by those in theart, assays that rely on decreases in such signals are also possible.

In one embodiment, assay reactions are performed “in situ” (alsoreferred to in various assay formats as “in vitro” and/or “ex vivo”depending on the sample), similar to FISH reactions. Since no exogeneousenzymes need be added, reagents can be added to cells (living,electroporated, fixed, etc.) such as histological samples for thedetermination of the presence of target sequences, particularly thoseassociated with disease states or other pathologies.

In addition, “in vitro” assays can be done where target sequences areextracted from samples. Samples can be processed (e.g. for paraffinembedded samples, the sample can be prepared), the reagents added andthe reaction allowed to proceed, with detection following as is done inthe art.

In some embodiments, ligation products (also referred to herein as“ligated products”) are detected using solid supports. For example, theligated products are attached to beads, using either anchorprobe/capture probe hybridization or other binding techniques, such asthe use of a binding partner pair (e.g. biotin and streptavidin). In oneembodiment, a transfer reaction results in a biotin moiety beingtransferred from the first ligation probe to a second ligation probecomprising a label. Beads comprising streptavidin are contacted with thesample, and the beads are examined for the presence of the label, forexample using FACS technologies.

In other embodiments, ligated products are detected using heterogeneousassays. That is, the reaction is done in solution and the product isadded to a solid support, such as an array or beads. Generally, oneligation probe comprises an anchor sequence or a binding pair partner(e.g. biotin, haptens, etc.) and the other comprises a label (e.g. afluorophore, a label probe binding sequence, etc.). The ligated productis added to the solid support, and the support optionally washed. Inthis embodiment, only the ligated product will be captured and belabeled.

In another aspect of the invention, one of oligonucleotide probes has anattached magnetic bead or some other label (biotin) that allows for easymanipulation of the ligated product. The magnetic bead or label can beattached to either the upstream or the downstream probe using any numberof configurations as outlined herein.

As described herein, secondary reactions can also be done, whereadditional functional moieties (e.g. anchor sequences, primers, labels,etc.) are added. Similarly, secondary amplification reactions can bedone as described herein.

Detection systems are known in the art, and include optical assays(including fluorescence and chemiluminescent assays), enzymatic assays,radiolabelling, surface plasmon resonance, magnetoresistance, cantileverdeflection, sequencing, surface plasmon resonance, etc. In someembodiments, the ligated product can be used in additional assaytechnologies, for example, as described in 2006/0068378, herebyincorporated by reference, the ligated product can serve as a linkerbetween light scattering particles such as colloids, resulting in acolor change in the presence of the ligated product.

In some embodiments, the detection system can be included within thesample collection tube; for example, blood collection devices can haveassays incorporated into the tubes or device to allow detection ofpathogens or diseases.

PCT applications WO 95/15971, PCT/US96/09769, PCT/US97/09739, PCTUS99/01705, WO96/40712 and WO98/20162, all of which are expresslyincorporated herein by reference in their entirety, describe novelcompositions comprising nucleic acids containing electron transfermoieties, including electrodes, which allow for novel detection methodsof nucleic acid hybridization.

One technology that has gained increased prominence involves the use ofDNA arrays (Marshall et al., Nat. Biotechnol. (1998) 16(1):27-31),especially for applications involving simultaneous measurement ofnumerous nucleic acid targets. DNA arrays are most often used for geneexpression monitoring where the relative concentration of 1 to 100,000nucleic acids targets (mRNA) is measured simultaneously. DNA arrays aresmall devices in which nucleic acid anchor probes are attached to asurface in a pattern that is distinct and known at the time ofmanufacture (Marshall et al., Nat. Biotechnol. (1998) 16(1):27-31) orcan be accurately deciphered at a later time such as is the case forbead arrays (Steemers et al., Nat. Biotechnol. (2000) 18(1):91-4; andYang et al., Genome Res. (2001) 11(11):1888-98.). After a series ofupstream processing steps, the sample of interest is brought intocontact with the DNA array, the nucleic acid targets in the samplehybridize to anchor oligonucleotides on the surface, and the identityand often concentration of the target nucleic acids in the sample aredetermined.

Many of the nucleic acid detection methods in current use havecharacteristics and/or limitations that hinder their broadapplicability. For example, in the case of DNA microarrays, prior tobringing a sample into contact with the microarray, there are usually aseries of processing steps that must be performed on the sample. Whilethese steps vary depending upon the manufacturer of the array and/or thetechnology that is used to read the array (fluorescence,electrochemistry, chemiluminescence, magnetoresistance, cantileverdeflection, surface plasmon resonance), these processing steps usuallyfall into some general categories: Nucleic acid isolation andpurification, enzymatic amplification, detectable label incorporation,and clean up post-amplification. Other common steps are sampleconcentration, amplified target fragmentation so as to reduce theaverage size of the nucleic acid target, and exonuclease digestion toconvert PCR amplified targets to a single stranded species.

The requirement of many upstream processing steps prior to contactingthe DNA array with the sample can significantly increase the time andcost of detecting a nucleic acid target(s) by these methods. It can alsohave significant implications on the quality of the data obtained. Forinstance, some amplification procedures are very sensitive to targetdegradation and perform poorly if the input nucleic acid material is notwell preserved (Foss et al., Diagn Mol. Pathol. (1994) 3(3):148-55).Technologies that can eliminate or reduce the number and/or complexityof the upstream processing steps could significantly reduce the cost andimprove the quality of results obtained from a DNA array test. Onemethod for reducing upstream processing steps involves using ligationreactions to increase signal strength and improve specificity.

As outlined above, the assays can be run in a variety of ways. In assaysthat utilize detection on solid supports, there are a variety of solidsupports, including arrays, that find use in the invention.

In some embodiments, solid supports such as beads find use in thepresent invention. For example, binding partner pairs (one on theligated product and one on the bead) can be used as outlined above toremove non-ligated reactants. In this embodiment, magnetic beads areparticularly preferred.

In some embodiments of the invention, capture probes are attached tosolid supports for detection. For example, capture probes can beattached to beads for subsequent analysis using any suitable technique,e.g. FACS. Similarly, bead arrays as described below may be used.

In one embodiment, the present invention provides arrays, each arraylocation comprising at a minimum a covalently attached nucleic acidprobe, generally referred to as a “capture probe”. By “array” herein ismeant a plurality of nucleic acid probes in an array format; the size ofthe array will depend on the composition and end use of the array.Arrays containing from about 2 different capture ligands to manythousands can be made. Generally, for electrode-based assays, the arraywill comprise from two to as many as 100,000 or more, depending on thesize of the electrodes, as well as the end use of the array. Preferredranges are from about 2 to about 10,000, with from about 5 to about 1000being preferred, and from about 10 to about 100 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single capture probe may be made as well. In addition, insome arrays, multiple substrates may be used, either of different oridentical compositions. Thus, for example, large arrays may comprise aplurality of smaller substrates. Nucleic acid arrays are known in theart, and can be classified in a number of ways; both ordered arrays(e.g. the ability to resolve chemistries at discrete sites), and randomarrays (e.g. bead arrays) are included. Ordered arrays include, but arenot limited to, those made using photolithography techniques (e.g.Affymetrix GeneChip®), spotting techniques (Synteni and others),printing techniques (Hewlett Packard and Rosetta), electrode arrays,three dimensional “gel pad” arrays and liquid arrays.

In a preferred embodiment, the arrays are present on a substrate. By“substrate” or “solid support” or other grammatical equivalents hereinis meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association ofnucleic acids. The substrate can comprise a wide variety of materials,as will be appreciated by those skilled in the art, including, but notlimited to glass, plastics, polymers, metals, metalloids, ceramics, andorganics. When the solid support is a bead, a wide variety of substratesare possible, including but not limited to magnetic materials, glass,silicon, dextrans, and plastics.

As will be appreciated, and as is also discussed above, a number ofdetection methods can be used to detect the presence of and quantify theligation products (or the amplicons of the ligation products) formed inaccordance with the present invention. Such detection methods can beutilized with any of the ligation products (or their amplicons) in anyof the assays discussed herein. Such detection methods include withoutlimitation: capillary electrophoresis, mass spectrometry, microarrayanalysis, sequencing, real-time PCR, optical detection, fluorescencedetection, bioluminescence detection, chemiluminescence detection,electrochemical detection, electrochemiluminescence detection andlateral flow detection.

In addition to the above described-assays, specific assays of use in theinvention include chemical ligation dependent probe amplification (CLPA)assays, which are discussed in further detail below.

Chemical Ligation Dependent Probe Amplification (CLPA)

In some aspects, the invention relates to chemical ligation dependentprobe amplification (CLPA) technology. CLPA is based on the chemicalligation of target specific ligation probes to form a ligation product.This ligation product subsequently serves as a template for an enzymaticamplification reaction to produce amplicons which are subsequentlyanalyzed using any suitable means, including without limitationdetection methods such as those discussed above: capillaryelectrophoresis, mass spectrometry, microarray analysis, sequencing,real-time PCR, optical detection, fluorescence detection,bioluminescence detection, chemiluminescence detection, electrochemicaldetection, electrochemiluminescence detection and lateral flowdetection. CLPA can be used for a variety of purposes including but notlimited to analysis of complex gene signature patterns. Unlike othertechniques, such as DASL (Bibikova, M., et al., American Journal ofPathology, (2004), 165:5, 1799-1807) and MLPA (Schouten, U.S. Pat. No.6,955,901), which utilize an enzymatic ligation reaction, CLPA uses achemical ligation reaction.

In one embodiment, the CLPA assay comprises the use of two or moreligation probes that incorporate reactive moieties that can self-ligatewhen properly positioned on a target sequence. In a preferredembodiment, a 3′-phosphorothioate moiety on a first probe reacts with a5′-DABSYL leaving group on a second probe (See Scheme I below).

The 5′-DABSYL group reacts about four times faster than other moieties,e.g. iodine, and also simplifies purification of the probes duringsynthesis.

CLPA has several distinct advantages over other sequence-basedhybridization techniques. First, CLPA can be applied directly to RNAanalysis without the need to make a DNA copy beforehand. Second, CLPA isrelatively insensitive to sample contaminants and can be applieddirectly to impure samples including body samples such as blood, urine,saliva and feces. Third, CLPA involves fewer steps than other knownmethods, thereby reducing the time required to gain a result. Moreover,CLPA probes can be stored dry, and properly designed systems willspontaneously react to join two or more oligonucleotides in the presenceof a complementary target sequence. Chemical ligation reactions showexcellent sequence selectivity and can be used to discriminate singlenucleotide polymorphisms.

Significantly, unlike enzymatic ligation methods, CLPA shows nearlyidentical reactivity on DNA and RNA targets which, as described morefully below, renders CLPA more efficient that other known systems, andexpands the scope of applications to which CLPA can be utilized.

Advantageously, the CLPA assay reduces the number of steps required todetect a target nucleic acid, which provides the potential to achieveresults in significantly shorter time periods. For example, the generalprocess flow for a standard reverse transcriptase (RT)-multiplexligase-dependent probe ligation (MLPA) involves the following steps:

-   -   1. Isolate total RNA.    -   2. Use Reverse Transcriptase to make cDNA copy.    -   3. Hybridize MLPA probe sets to the cDNA target overnight.    -   4. Add DNA Ligase to join target-bound probes.    -   5. Amplify ligated probes, e.g. PCR amplification using Taq        polymerase and fluorescently labeled PCR primers.    -   6. Analyze the sample, for example, by CE.

Unlike standard RT-MLPA, CLPA enables analysis to be carried outdirectly on cell and blood lysates and on RNA targets without a need forsteps involving isolating the RNA and performing a reverse transcriptionreaction to make a cDNA copy prior to ligation. This shortens the timefor achieving a result and provides a means to achieve faster analysis.

In further embodiments, CLPA methods of the present invention areconducted on formalin fixed paraffin embedded (FFPE) tissues. In stillfurther embodiments, the FFPE tissues are first subjected to a chemicaldenaturant to lyse the tissues, and then are subjected to any of theCLPA methods described herein. In yet further embodiments, proteinase Kand/or sonication are used to further treat the FFPE tissues prior toapplication of CLPA methods.

In further embodiments of the invention, faster reaction times arefurther facilitated by driving the hybridization reaction with higherprobe concentrations. Thus, for example, input probe sets may beincorporated in the CLPA reaction at relatively high concentrations, forexample, approximately 100-fold higher than those typically used in anMLPA reaction. Elevating the probe concentration significantly reducesthe time required for the hybridization step, typically from overnightto between about 15 minutes to about 1 hour.

CLPA probes are generally incorporated in a reaction at a concentrationof 250 nanomolar (nM) to 0.01 pM (picomolar) for each probe. Generally,the concentration is between about 1 nM to about 1 pM. Factors toconsider when choosing probe concentration include the particular assayand the target being analyzed. The S- or phosphorothioate or Nucleophileprobe and L- or leaving group or DABSYL containing probes areincorporated at a concentration that equals or exceeds the concentrationof the target. Total concentration of S- and L-probes can reach as highas 10 micromolar (uM). As a non-limiting example, 1 nM for each S and Lprobe×250 CLPA probe pairs would equal 500 nm (1 nm per probe×2 probesper pair×250 targets) at 10 nM for each probe would mean a totalconcentration of 5 uM.

The target concentration usually ranges from about 10 micrograms oftotal RNA to about 10 nanograms, but it can be a little as a single copyof a gene.

When higher probe concentrations are used it is generally preferred toincorporate a purification step prior to amplification, especially forhigh multiplex analysis (e.g. greater than about 5 targets). In oneembodiment of this aspect of this invention, a solid support basedcapture methodology can be employed including membrane capture, magneticbead capture and/or particle capture. In a preferred embodiment, abiotin/streptavidin magnetic bead purification protocol is employedafter ligation and prior to enzymatic amplification. In some instances,the magnetic particles can be directly added to the amplification mastermix without interfering with the subsequent amplification reaction. Inother instances, it is preferable to release the capturedoligonucleotide from the beads and the released oligonucleotide solutionis subsequently amplified without the capture particle or surface beingpresent.

In a preferred embodiment, CLPA involves hybridization of a set ofprobes to a nucleic acid target sequences such that the probes canundergo self-ligation without addition of a ligase. After a ligationproduct is produced, amplification is generally used to facilitatedetection and analysis of the product. For this purpose, probes aredesigned to incorporate PCR primers such as, e.g. universal PCR primers.In a preferred embodiment, the universal primers are not added untilafter the ligation portion of the reaction is complete, and the primersare added after surface capture purification (such as with the use oftarget capture probes, which are described in further detail herein)along with the polymerase, often as part of a PCR master mix.

The CLPA probes possess reactive moieties positioned such that when theCLPA probes are bound to the nucleic acid target, the reactive moietiesare in close spatial orientation and able to undergo a ligation reactionwithout the addition of ligase enzyme. In a preferred embodiment, thechemical ligation moieties are chosen so as to yield a ligated reactionproduct that can be efficiently amplified by an amplification enzyme,which is in preferred embodiments a DNA polymerase, but can includeother enzymes such as RNA polymerase. Without being bound by theory,chemical ligation chemistries and probe set designs that producereaction products that more closely resemble substrates that are knownas being able to be amplified by DNA and RNA polymerases are more likelyto yield efficient probe sets that can be used in the CLPA assay.Especially preferred reaction chemistries are chemical moieties thatyield reaction products that closely resemble native DNA such asillustrated in Scheme 1 involving a reaction between a3′-phosphorothioate and a 5′ DABSYL leaving group. In another preferredembodiment, probes sets comprise a 3′-diphosphorothioate (Miller, G. P.et al, Bioorganic and Medicinal Chemistry, (2008) 16:56-64, which ishereby incorporated by reference in its entirety and in particular forall teachings related to probes and 3′-diphosphorothioate chemistries)and a 5′-DABSYL leaving group.

The CLPA probes of the present invention may also incorporate a stuffersequence (also referred to herein as a variable spacer sequence) toadjust the length of the ligation product. The length of the stuffersequence can be specific to the target sequence to which the ligationprobe is directed, thus providing a convenient means to facilitateanalysis of ligation product(s). The stuffer sequence can be located oneither or both probes. In embodiments utilizing a 3′-phosphorothioateprobe, the stuffer sequence is in preferred embodiments incorporated onthis probe (also referred to herein as the “S probe”). As is alsodiscussed above, the stuffer sequence may be located in a region of theligation probe that is non-complementary to the target nucleic acid, orthe stuffer sequence may be located in a region that at least partiallyoverlaps with a region that is complementary to a domain of the targetnucleic acid. In further embodiments, the stuffer sequence is locatedbetween the primer sequence and the probe domain.

In further embodiments of the invention, CLPA-CE, the stuffer sequenceis varied in length in order to produce one or more variable lengthligation products which provide the basis for detection andidentification of specific target sequences based on length variation.In a preferred embodiment, variable length ligation products areanalyzed by capillary electrophoresis (CE). Generally, stuffer sequencesare included such that the length of different ligation products variesin a range of at least 1 base pair to about 10 base pairs; preferablyfrom 1 base pair to 4 base pairs. In a preferred embodiment, the lengthof the different ligation products vary from approximately 80 bp toabout 400 bp; preferably in a range of about 100 bp to about 300 bp;more preferably in a range of about 100 bp to about 200 bp. In furtherembodiments, the stuffer sequences are of a length such that the lengthof the ligation product is from about 5-1000, 10-950, 15-900, 20-850,25-800, 30-750, 35-700, 40-650, 50-600, 55-550, 60-500, 65-450, 70-400,75-350, 80-300, 85-250, 90-200, 95-150 base pairs.

In some embodiments, CLPA probes may further contain other optionalelement(s) to facilitate analysis and detection of a ligated product.Such elements include any of the elements discussed above in the sectionon ligation probe pairs. For example, for embodiments referred to hereinas CLPA-MDM (and discussed in further detail below), it is preferredthat at least one of the ligation probes for incorporate an arraybinding sequence to bind to an appropriate capture sequence on amicroarray platform. For CLPA-MDM, the different CLPA reaction productsare not separated by size differences but by the differences in thearray binding sequence. In this embodiment, the sequence of the arraybinding sequence is varied so that each CLPA probe will bind to a uniquesite on a DNA microarray. The length of the array binding sequence inCLPA-MDM usually varies from 15 to 150 bases, more specifically from 20to 80 bases, and most specifically from 25 to 50 bases.

In further embodiments, CLPA probes preferably also include otherelements to facilitate purification and/or analysis including but notlimited to labels such as fluorescent labels and hapten moieties suchas, for example, biotin for purifying or detecting ligation product(s).For example, probes and/or ligation product(s) that incorporate biotincan be purified on any suitable avidin/streptavidin platform includingbeads. While biotin/avidin capture systems are preferred, other haptensystems (e.g. Digoxigenin (DIG) labeling) can be used, as canhybridization/oligonucleotide capture. Hybridization/oligonucleotidecapture is a preferred method when it is desirable to release thecapture product from the beads at a later stage. In addition to magneticbeads, anti-hapten labeled supports (filter paper, porous filters,surface capture) can be used.

CLPA probe-labeling can be on either probe, either at the end orinternally. Preferably biotin is incorporated at the 5′ end on thephosphorothioate (S-probe).

In a preferred embodiment, a CLPA probe set consists of 2oligonucleotide probes with complementary reactive groups (FIGS. 1 and2). In another embodiment, the CLPA probe set may consist of 3 or moreprobes that bind adjacent to each other on a target. In a preferredembodiment of the 3-probe CLPA reaction, the outer probes are designedto contain the enzymatic amplification primer binding sites, and theinner probe is designed to span the region of the target between theother probes. In a more preferred embodiment, the outer probes havenon-complementary reactive groups such that they are unable to reactwith each other in the absence of the internal (middle) probe (FIG. 3).In some instances, both outer probes may have similar reactive moietiesexcept that one group is at the 5′ end of one probe and the 3′-end ofthe other probe, and the L-probe chemistries may also be similar to eachother except for positioning on the probe. As is known to one who isskilled in the art, different chemical reagents and processes may beneeded to manufacture the probes for the 3-probe CLPA reaction comparedto the probes for the 2-probe CLPA system.

In a preferred embodiment of the 3-probe CLPA system, one outer probecontains a 3′ phosphorothioate (3′S-probe), the other outer probecontains a 5′-phosphorothioate (5′-S-probe) and the center probecontains both a 3′- and a 5′-DABSYL leaving group. The manufacture of a5′-DABSYL leaving group probes has been reported previously (Sando etal, J. Am. Chem. Soc., (2002), 124(10) 2096-2097). We recently developeda new DNA synthesis reagent that allows for the routine incorporation ofa 3′-DABSYL leaving group (see FIG. 4).

In further embodiments and in accordance with any of the CLPA methodsdescribed herein, the assays may be conducted in the presence of abuffer of the invention. Such buffers are described in detail above, andany combination of buffer components may be of use in the CLPA methodsof the invention. In preferred embodiments, CLPA methods of theinvention, including the target capture CLPA, CLPA-CE, and CLPA-MDMmethods described below, are conducted in a buffer of the invention,including buffers comprising a denaturant comprising a chaotropic cationsuch as guanidinium hydrochloride.

Target Capture CLPA

In further embodiments, CLPA assays utilizing two or more ligationprobes can be combined with a target capture probe that binds upstreamor downstream to the CLPA probe set on the same nucleic acid target.This target capture probe contains a capture moiety (which can withoutlimitation include a hapten, a bead, an oligonucleotide capturesequence, and so forth) that can be used to selectively capturecomplexes/molecules that are bound to the target capture throughhybridization or other interactions (FIG. 10).

The target capture probe can be used with any embodiment of CLPAdiscussed herein.

As is also discussed in further detail above, more than one targetcapture probe can be applied to a target nucleic acid. These multipletarget capture probes can be designed to flank one or more ligationproducts, either by binding at or near both the 3′ and 5′ ends of thetarget nucleic acids or by interleaving the target capture probesbetween multiple ligation products. As is discussed in further detailbelow, multiple target capture probes can be used in methods forassessing the quality/integrity of target nucleic acids, particularlyRNA.

CLPA-CE

In one aspect, the present invention provides methods for using ligationprobes containing variable spacer sequences to produce ligationproducts, and those ligation products are then detected using capillaryelectrophoresis.

In certain embodiments, the ligation products are first amplified usingany method known in the art (including PCR) to produce amplicons, and itis then the amplicons that are detected using capillary electrophoresis.In further embodiments, the ligation products and/or amplicons aredetected by size differentiation capillary electrophoresis (CE) on asieving matrix, or by slab gel electrophoresis.

A schematic representation for CLPA-CE is provided in FIG. 1. In theembodiment depicted in this figure, a blood sample is subjected to celllysis by any appropriate means, including without limitation chemical,mechanical or osmotic cell lysis. Preferably, chemical lysis is used.

Following chemical lysis, probes directed to target nucleic acids areapplied. FIG. 6 provides a general schematic representation of thedesign of a probe set for CLPA-CE analysis. In this example, the S probeis designed to include a universal PCR primer for subsequentamplification of ligation product(s). The S probe also includes astuffer sequence designed with a length that correlates with a specifictarget sequence. The S probe also includes a target binding sequence(also referred to herein as a “probe domain”) that is complementary to atarget domain of the target nucleic acid comprising the target sequenceto which the stuffer sequence correlates. As will be appreciated, a setof probes may contain a plurality of S probes that are all directed tothe same target domain, or the set may contain a mixture of S probesdirected to different target domains. Likewise, the L-probe includes atarget binding sequence that is complementary to a target domainadjacent to the target domain to which the S probe binds. In thisembodiments, the L-probe also includes a universal primer. In furtherembodiments, one or both of the probes are labeled with a fluorophore(FAM, Cy3, Cy5, etc), however they can also be detected withoutfluorescence labeling. The labeling is in some embodiments accomplishedby using a fluorescently labeled PCR primer.

In the embodiment of CLPA-CE probes pictured in FIG. 6, the S probe alsoincludes a biotin moiety at the 5′ end to facilitate purification andremoval of unligated probe.

As shown in FIG. 1, after the S- and L-probes are hybridized to thetarget nucleic acid, the probes undergo a spontaneous chemical ligationto produce a ligation product. In some embodiments in accordance withFIG. 1, a bead purification step is used to remove unligated probes,although the removal of unligated probes is not a required step for allembodiments of CLPA-CE methods. After ligation and the optional removalof unligated probes, the ligation products are amplified. In embodimentsin which the ligation probes contain universal primer sequences,universal PCR primer pairs are used to produce amplicons. In embodimentsthat utilize other types of primer sequences, complementary primer pairscan be used to generate amplicons.

Due to the presence of the variable spacer sequence in one (or in someembodiments both) of the ligation probes, the amplicons have a uniquelength that corresponds to a specific target nucleic acid. Thoseamplicons are then analyzed using capillary electrophoresis analysis,and the relative intensity of each peak corresponds to relativeexpression. (FIG. 1 and FIG. 7). In alternative embodiments, othersuitable size separation techniques can be used to determine targetnucleic acid expression in the sample.

In further embodiments and in accordance with any of the above, theCLPA-CE method includes a step of hybridizing a target capture probe tothe target nucleic acid. The resulting target complex comprising thetarget capture probe, the target nucleic acid and the ligation productis then, prior to amplification of the ligation product, captured on asurface or a substrate through the capture moiety of the target captureprobe. In further embodiments, unbound target nucleic acids and ligationprobes are removed, leaving only the target complexes and the ligationproducts. The ligation products can then be amplified and analyzedaccording to any of the methods described herein. In some embodiments,the ligation products are removed from the target complex prior toamplification using methods known in the art, including heating and theaddition of denaturants or other agents to change the hybridizationconditions.

CLPA-MDM

In another embodiment of this aspect of the invention, CLPA ligationproducts are analyzed/detected by microarray analysis (CLPA-MDM). Aschematic representation of CLPA-MDM is provided in FIG. 2. CLPA-MDMdiffers from CLPA-CE in at least the following respects. First, theprobe sets differ in design. For example, a general representation of aCLPA-MDM probe set is depicted in FIG. 2. As with CLPA-CE probes,CLPA-MDM probe sets can include universal primers for amplification ofligation product(s). They also include target specific sequences, aswell as ligation moieties for enzyme-independent ligation. Additionally,CLPA-MDM probes also may include a stuffer sequence, however the purposeof this stuffer sequence is to adjust the size of the CLPA-MDM to thesame length in an effort to standardize enzymatic amplificationefficiency. Normalization of amplicon size is not a requirement but canprovide advantages for amplification efficiency. A second differencebetween the design of CLPA-CE and CLPA-MDM probe sets is that the latterinclude a unique array binding sequence for use with an appropriatemicroarray platform.

In respect of the CLPA-MDM aspect of the invention, a microarray bindingsite (ABS sequence) is incorporated into the probe designs for use witha “universal” microarray platform for the detection. Similar to theCLPA-CE system, probes are preferably labeled with a fluorophore, forexample by using a fluorescently labeled PCR primer. Alternatively, forexample, a sandwich assay labeling technique can be used for the finalread-out. Sandwich assays involve designing the probes with a common(generic) label binding site (LBS) in place or in addition to thestuffer sequence and using a secondary probe that will bind to this siteduring the array hybridization step. This methodology is particularlyuseful when it is desirable to label the arrays with a chemiluminescentsystem like a horse radish peroxidase (HRP) labeled oligonucleotide, orwith an electrochemical detection system. Generally, planar microarraysare employed (e.g. microarrays spotted on glass slides or circuit board)for the read-out. However, bead microarrays such as those available fromLuminex and Illumine can also be used (e.g. Luminex xMAP/xtag).

Nucleic Acid Quality Assessment

In certain aspects, multiple CLPA probe sets that differ in bindingposition on a nucleic acid target relative to a target capture probe areused (FIG. 11) in methods to assess nucleic acid quality. In theembodiment pictured in FIG. 11, a target capture probe is hybridized toone end of the target nucleic acid, and multiple ligation productsproduced from different probe sets directed to different target domainsof the target nucleic acid are used to produce a target complexcomprising the target capture probe and the multiple ligation products.

In such aspects, differences in the signals from the different ligationproducts provide an assessment of the quality of the nucleic acidtarget. For example, by measuring the differences in captureefficiency/signal from the unique CLPA probe set located closest to thetarget capture probe (e.g., 1-100 bp away from the target capture probe)compared to a CLPA probe set further from the target capture probe(e.g., 300-1000 bp away), it is possible to infer the fragmentation orlevel of degradation of the nucleic acid target. In further exemplaryembodiments, the relative ratio of signals generated for the differentCLPA probe sets provides a measure of the level of degradation for aparticular nucleic acid target. In further embodiments, the ligationprobe sets are designed such that they are directed to target domains atknown distances from the domain to which the target capture probe ishybridized. In such embodiments, the target complex comprising thetarget capture probe, the ligation products and the target nucleic acidare captured on a surface or a substrate through a capture moiety on thetarget capture probe, and unbound target nucleic acids and ligationprobes are separated from the captured target complexes. The relativeratio of signals associated with the multiple different probe sets thenprovides an indication of the amount of degradation present in thetarget nucleic acids in the sample, and the known distances between theligation products and the target capture probe provides a kind of“molecular ruler” for quantifying the degradation in the target nucleicacids. For example, in reference to FIG. 11, if in an exemplaryembodiment the majority of the target nucleic acids in a sample aredegraded in a domain located between the domains to which CLPA set 2 andCLPA set 3 are hybridized, then the signal from CLPA sets 1 and 2 willbe relatively larger than the signal from CLPA set 3. This relativeratio between the signals provides an indication of the length andintegrity of the target nucleic acids in the sample.

As will be appreciated by those in the art, the spacing of the differentprobe sets will depend on the size of the original target and thedesired information. The spacing can be relatively equidistant (e.g. theuse of 3 probe sets spaced roughly 30% of the total length apart), orcan be clustered as desired.

In further embodiments, multiple target capture probes are used forassessment of the quality of the nucleic acid target. As is furtherdiscussed herein, the different target capture probes can be designed tohybridize near or at the ends of the target nucleic acid and may also orin the alternative be designed to hybridize between different ligationproducts. In some embodiments, two target capture probes are designed tohybridize to domains both upstream and downstream of one or moreligation products. Use of the two different capture probes can helpensure that even in highly degraded samples, all target complexescomprising ligation products are efficiently captured on a surface or asubstrate and separated from unbound target nucleic acids and ligationprobes prior to amplification of the ligation products.

Information on degradation is useful in assessing the quality of thenucleic acid and more specifically RNA contained in samples. Thistechnique is particularly useful in assessing the quality of RNA informalin fixed paraffin embedded (FFPE) tissue samples and other sampleswhere the risk of sample degradation is high.

As discussed above, the difference in capture efficiency can beindirectly determined by measuring the relative ratio of signals. Thismethodology can be combined with microarray, CE, sequencing, real timePCR and other methods of nucleic acid analysis to further assess thetarget nucleic acids in accordance with any of the methods describedabove.

VIII. Hardware

In one aspect of the invention, a fluidic device similar to thosedescribed by Liu (2006) is used to automate the methodology described inthis invention. See for example U.S. Pat. No. 6,942,771, hereinincorporated by reference for components including but not limited tocartridges, devices, pumps, wells, reaction chambers, and detectionchambers. The fluidic device may also include zones for capture ofmagnetic particles, separation filters and resins, including membranesfor cell separation (i.e. Leukotrap™ from Pall). The device may includedetection chambers for in-cartridge imaging of fluorescence signalgenerated during Real-Time PCR amplification (i.e. SYBR green, Taqman,Molecular Beacons), as well as capillary electrophoresis channels foron-device separation and detection of reactions products (amplicons andligation products). In a preferred embodiment, the capillaryelectrophoresis channel can be molded in a plastic substrate and filledwith a sieving polymer matrix (POP-7™ from Applied Biosystems). Channelscontaining non-sieving matrix can also be used with properly designedprobe sets.

In a preferred embodiment, the devices of the invention comprise liquidhandling components, including components for loading and unloadingfluids at each station or sets of stations. The liquid handling systemscan include robotic systems comprising any number of components. Inaddition, any or all of the steps outlined herein may be automated;thus, for example, the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates; holderswith cartridges and/or caps; automated lid or cap handlers to remove andreplace lids for wells on non-cross contamination plates; tip assembliesfor sample distribution with disposable tips; washable tip assembliesfor sample distribution; 96 well loading blocks; cooled reagent racks;microtiter plate pipette positions (optionally cooled); stacking towersfor plates and tips; and computer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradeable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 0° C. to 100° C.; this is in addition to orin place of the station thermocontrollers.

In a preferred embodiment, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, the instrumentation will include a detector, whichcan be a wide variety of different detectors, depending on the labelsand assay. In a preferred embodiment, useful detectors include amicroscope(s) with multiple channels of fluorescence; plate readers toprovide fluorescent, electrochemical and/or electrical impedanceanalyzers, ultraviolet and visible spectrophotometric detection withsingle and dual wavelength endpoint and kinetics capability,fluorescence resonance energy transfer (FRET), luminescence, quenching,two-photon excitation, and intensity redistribution; CCD cameras tocapture and transform data and images into quantifiable formats;capillary electrophoresis systems, mass spectrometers and a computerworkstation.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. Again, as outlined below, this may be in addition to orin place of the CPU for the multiplexing devices of the invention. Thegeneral interaction between a central processing unit, a memory,input/output devices, and a bus is known in the art. Thus, a variety ofdifferent procedures, depending on the experiments to be run, are storedin the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

IX. Overall Process

One distinct advantage of aspects of the present invention is theability to collect samples, particularly for RNA detection, into astabilization buffer that allows both for a) long time periods beforetesting without sample degradation (of particular use in the assay ofRNA, as RNA is degraded extremely rapidly under traditional conditions)and b) the ability to run the assay in the collection buffer withouthaving to do further purification steps to remove components from themixture that prevent enzymatic assays from being run, such as highdenaturing salt concentrations. The ability to collect samples such asblood directly into a buffer that simultaneously lyses the cells andstabilizes the RNA for long periods of time (e.g. days to weeks orlonger) allows remote collection and a time lag before assay processing.In addition, these methods thus avoid any special conditions forhandling, such as avoidance of heat exposure and/or cold chain handling.For example, samples can be collected and mailed using regular mail andbe tested without additional steps.

Furthermore, the ability to assay directly in the collected sample is asignificant benefit over the use of sample preparation steps to purifythe samples for traditional enzyme based assays.

In further embodiments, a sample is collected into a stabilizationbuffer in one geographic location and can then be transported to adifferent geographic location prior to conducting assays on the samplein accordance with any of the aspects and embodiments of the inventiondiscussed herein. In other words, as is discussed above, the presentinvention provides methods and compositions that result in a stabilizedsample that can be collected in one geographic location and thensubjected to chemical ligation methods and assays as discussed herein ina different geographic location.

X. Kits

In another aspect of the invention, a kit for the routine detection of apredetermined set of nucleic acid targets is produced that utilizesprobes, techniques, methods, and a chemical ligation reaction asdescribed herein as part of the detection process. The kit can compriseprobes, target sequences, instructions, buffers, and/or other assaycomponents.

In a further aspect the invention provides for kits for stabilizing anddetecting or quantifying RNA in a sample comprising a buffer solutioncomprising a denaturant, a first ligation probe, and a second ligationprobe. In a preferred embodiment, the denaturant is chosen from thegroup consisting of guanidinium hydrochloride and guanidiniumisothiocyanate. In a further preferred embodiment the buffer solutionfurther comprises EDTA, a reducing agent, a surfactant and a pH buffer.In particularly preferred embodiment, the reducing agent is chosen fromthe group consisting of DTT and mercaptoethanol. In another particularlypreferred embodiment, the surfactant is chosen from the group consistingof Triton X-100 and sodium N-lauroylsarcosine.

In still further aspects, the present invention provides kits fordetecting a target nucleic acid sequence where that target sequencecomprises an adjacent first and second target domain. Such kits mayinclude: a 2× lysis buffer comprising 6 M GuHCl and at least one set ofligation probes, where that at least one set of ligation probes includesa first ligation probe that includes a first probe domain complementaryto the first target domain of the target nucleic acid sequence and asecond ligation probe that includes a second probe domain complementaryto the second target domain of the target nucleic acid sequence. As isfurther discussed above, the ligation probes included in kits of theinvention may include further functionalities, including stuffersequences, primer sequences, and anchor sequences, and any combinationthereof.

EXAMPLES Example 1: Quantitative Multiplex Detection of Five Targets

Multiplex CLPA reactions were performed using five (5) DNA target mimics(corresponding to portions of the MOAP1 (SEQ ID NO:5), PCNA (SEQ IDNO:9), DDB2 (SEQ ID NO:12), BBC3 (SEQ ID NO:16) and BAX(SEQ ID NO:19)genes) combined in one reaction in the presence of their respective CLPAprobes (Table 1) (S and L probes at 1 nM each). The target mimics werepooled in different concentration as shown in Table 2. The targetmimics, S probes and L probes were incubated in PCR buffer (1×PCR bufferis 1.5 mM MgCL₂, 50 mM KCl, 10 mM Tris-HCl pH 8.3) for 1 hour at 50° C.A 1 μl aliquot of each reaction mixture was used as template for PCRamplification using Dynamo SYBR green PCR mix in the presence ofUniversal Primers (SEQ ID NOS 1 and 2, 300 nM). The samples were PCRcycled for 27 cycles (95° C. 15 min followed by 27 cycles of 95° C. (10s), 60° C. (24 s), 72° C. (10 s). After PCR amplification, the sampleswere denatured and injected into an ABI 3130 DNA sequencer (capillaryelectrophoresis instrument). The CE trace from the ABI for the 3 samplesas well as a plot of the peak versus target mimic concentration of PCNAis shown in FIG. 7 and a plot of the linear response of the signal ofPCNA as a function on input concentration is shown in FIG. 8.

TABLE 1  Probe and target sequence information. SEQ Amplicon ID NameSequence Detail Size  1 Forward PCR Primer FAM-GGGTTCCCTAAGGGTTGGA  2Reverse PCR Primer GTGCCAGCAAGATCCAATCTAGA  3 MOAP1-LLTACATCCTTCCTAGTCAATTACACTCTAGATTGGATCTTGCTGGCAC  47  4 MOAP1-S5′-Biotin-  41 GGGTTCCCTAAGGGTTGGATAGGTAAATGGCAGTGTAGAACSLigated MOAP1 Amplicon  88  5 MOAP1-TargetGTGTAATTGACTAGGAAGGATGTAGTTCTACACTGCCATTTACCTA mimic  6 MOAP1-RNA TargetGUGUAAUUGACUAGGAAGGAUGUAGUUCUACACUGCCAUUUACCUA mimic  7 PCNA-LLTGGTTTGGTGCTTCAAATACTCTCTAGATTGGATCTTGCTGGCAC  45  8 PCNA-S Biotin-  63GGGTTCCCTAAGGGTTGGATCGAGTCTACAGATCC CCAACTTTCATAGTCTGAAACTTTCTCCSLigated PCNA Amplicon 108  9 PCNA-Target MimicAGTATTTGAAGCACCAAACCAGGAGAAAGTTTCAGACTATGA 10 DDB2-LLTAGCAGACACATCCAGGCTCTAGATTGGATCTTGCTGGCAC  51 11 DDB2-S Biotin-  49GGGTTCCCTAAGGGTTGGATCGAGTCTACTCCAAC TTTGACCACCATTCGGCTACSLigated DDB2 Amplicon  96 12 DDB2 Target MimicGCCTGGATGTGTCTGCTAGTAGCCGAATGGTGGTCA 13 DDB2-RNA TargetGCCUGGAUGUGUCUGCUAGUAGCCGAAUGGUGGUCA Mimic 14 BBC3-LLTCCGAGATTTCCCCCTCTAGATTGGATCTTGCTGGCAC  38 15 BBC3-S Biotin-  37GGGTTCCCTAAGGGTTGGATCCCAGACTCCTCCCTCTS Ligated BBC3 Amplicon  75 16BBC3 Target Mimic GGG GGA AAT CTC GGA AGA GGG AGG AGT CTG GG 17 BAX-LLTCACGGTCTGCCACGCTCTAGATTGGATCTTGCTGGCAC  39 18 BAX-SBiotin-GGGTTCCCTAAGGGTTGGA TGA GTC TAC  53 ATGA TC CT TCCCGCCACAAAGATGGSLigated BAX Amplicon  92 19 BAX-Target MimicCGTGGCAGACCGTGACCATCTTTGTGGCGGGA 20 3-phosphorothioate Biotin-  51 GAPDHGGGTTCCCTAAGGGTTGGACGGACGCCTGCTTCAC CACCTTCTTGATGTCAS 21 Middle 2L probeLTCATATTTGGCAGGTTTTTCTAGACGGCAGGTL  32 GAPDH 22 5′-phosphorothioateSCAGGTCCACCACTGACACGTTGGCAGTTCTAGAT  50 GAPDH TGGATCTTGCTGGCACLigated 3-probe amplicon 133 23 ReversePrimerBiotin-ATTAACCCTCACTAAAGGGA A3632-p 24 GAPDH TargetACT GCC AAC GTG TCA GTG GTG GAC CTG ACC MimicTGC CGT CTA GAA AAA CCT GCC AAA TAT GATGAC ATC AAG AAG GTG GTG AAG CAG GCG TC 25 GAPDH 3-LLTTTTCTAGACGGCAGGTCAGGTCCACCAGATGAT CGACGAGACACTCTCGCCATCTAGATTGGATCTTGCTGGCAC 26 GAPDH 3-S GGGTTCCCTAAGGGTTGGACGGACCAACTCCTCGCCATATCATCTGTACACCTTCTTGATGTCATCATATT TGGCAGGTS 27 GAPDH-3-(FAM)ccaactcctcgccatatcatctgtacaccttcttg FAM/BHQ-1 (BHQ-1) Taqman Probe28 GAPDH 4-L LTGCTGATGATCTTGAGGCTGTTGTCATACTGATGATCGACGAGACACTCTCGCCATCTAGATTGGATCT TGCTGGCAC 29 GAPDH-4-SGGGTTCCCTAAGGGTTGGACGATGGAGTTGATGCT GACGGAAGTCATAGTAAGCAGTTGGTGGTGCAGGAGGCATS 30 GAPDH-4-QUASAR (Quasar 670)tgctgacggaagtcatagtaagcagttggt670/BHQ-2 Taqman (BHQ-2) Probe 31 PCNA 2-LLTCCTTGAGTGCCTCCAACACCTTCTTGAGGATGAT CGACGAGACACTCTCGCCATCTAGATTGGATCTTGCTGGCAC 32 PCNA 2-S GGGTTCCCTAAGGGTTGGACGGTACAACAAGACCCAGCTGACGACTCTTAATATCCCAGCAGGCCTCGTT GATGAGGS 33 PCNA 2-Cal Fluor(CAL Red 610)ctgacgactcttaatatcccagcaggcctcgtt Orange 560/BHQ-1 (BHQ-2)34 DDB2-2-L LTTAGTTCCAAGATAACCTTGGTTCCAGGCTGATGATCGACGAGACACTCTCGCCATCTAGATTGGATCT TGCTGGCAC 35 DDB2-2-SBiotinGGGTTCCCTAAGGGTTGGACGTTAGACGCCAATAGGAGTTTCACTGGTGGCTACCACCCACTGAGA GGAGAAAAGTCATS 36 DDB2-2-(CAL Fluor(Cal Orange 560)cgccaataggagtttcactggtggctacca Orange 560/BHQ-1 (BHQ-2)37 MRPS5-TC [BIOTIN]GCCAGAGAGGTTACGTGGCGGCT  30 CTCTTCA 38 MRPS5-SGGATGCTATGAGCGATCTGCAGCGTGCAG  58 TCTTCACATCTTCCCAGTCCAGTTTGACGS 39MRPS5-L LTCTGGAACCTCATCTTCTGGCTCTGGATC  79 CTTCCTAAGTGAATGTTGACAGGATGCTCTAGATTGGATCTTGCTGGCAC Ligated MRPS5 Amplicon 137 40 PCNA-TC[BIOTIN]TCTTCATCCTCGATCTTGGGAGCC  30 AAGTAG 41 PCNA-SGGATGCTATGAGCGATCTGCAGCCACTAT  76 ACATCTTACTATACTTTACTCTACAACAAGGGGTACATCTGCAGACAS 42 PCNA-L LTACTGAGTGTCACCGTTGAAGAGAGTGG  70AGTGGCTTTTGTAAAGTCTTCTAGATTGG ATCTTGCTGGCAC Ligated PCNA Amplicon 146 43CDR2-TC [BIOTIN]AGAGTGATCGGTATTTTGTTCTCT  29 GTTCA 44 CDR2-SGGATGCTATGAGCGATCTGCAGCGCAATT  86 CATTTCATTCACAATCAATCTAAAGATCTCCTTAAACAACGCTTTGTATTCTGGAGGS 45 CDR2-L LTGTTGTAGGGGAACTCACGGGCTCTGGG101 TTGACAGAGGCCAGTTAGGATGTTACCAC CAGTGAATGTTGACAGGATCCTCTAGATTGGATCTTGCTGGCAC Ligated CDR2 Amplicon 187 *Luminex Bead # 46 GAPDH-LLTCCATTGATGACAAGCTTCCCGTTCTCAGCTCGCGTTCTAAGC TTCCCTTTAGTGAGGGTTAAT 47GAPDH-S Biotin-  12 TAATACGACTCACTATAGGGCGAGTAGAAAGTTGAAATTGATTATGATCTCGCTCCTGGAAGATGGTGATGGGATTS 48 ACTG2-LLTTCTCCCAGTGACTGAGGGCTGGTGTGTCTTTGGCTCCCTTTA GTGAGGGTTAAT 49 ACTG2-SBiotin-  72 TAATACGACTCACTATAGGGCGAATTGAGAAAGAGATAAATGATAGGGACTGGAGCACCGAGGGTATGAGAGGTTCS 50 ACPP-LLTTCAACTCCTTGGCTAGTACACTTCGGTCTAGCGCTCCCTTTA GTGAGGGTTAAT 51 ACPP-SBiotin-  66 TAATACGACTCACTATAGGGCGGTAAGAGTATTGAAATTAGTAAGAGGTCTCCATGCCGAAACACCAAAGTCACAAACS 52 RDH11-LLTGTGCATCTCAAAGCCATCTGCTGTCTTCGGCTCCCTTTAGTG AGGGTTAAT 53 RDH11-SBiotin-  63 TAATACGACTCACTATAGGGCGTTTGTTGTTAAGTATGTGATTTAGGGAGGAAGTGACCCAAGTGGTTGACTCCTAS 54 DES-LLTGTTCTCTGCTTCTTCCTTCAACTGAATCTCCTCCTGCTTCCCTT TAGTGAGGGTTAAT 55 DES-SBiotin-  68 TAATACGACTCACTATAGGGTATTTGATAAGAGAATGAAGAAGTATCCACGTCCGCTCGGAAGGCAGCCAAATS 56 Primer A3534-pp-TAATACGACTCACTATAGGG *Luminex Product Insert Sheet MagTAG-PlexMicrospheres. L = DABSYL ligation moiety S = phosphorothioate moiety

TABLE 2 Sample Concentrations Sample Target Mimic Concentrations 1 AllTarget mimics at 10 pM final Concentration 2 MOAP1, DDB2 and BBC3 at 10pM, PCNA at 5 pM and BAX at 2 pM 3 MOAP1, DDB2 and BBC3 at 10 pM, PCNAat 1 pM and BAX at 0.5 pM

Example 2: CLPA Reactions Using MOAP1 and DDB2 DNA and RNA Target Mimics

Reactions were prepared in duplicate as presented in Table 3 using DNAor RNA target mimics for the MOAP1 and DDB2 genes and CLPA probes setsdesigned to target the sequences. The probe numbers refer to the SEQ IDNOs in Table 1. The reagents were added in the concentrations andvolumes shown in Table 4. The respective S-probe, L-probe and targetmimic were heated to 50° C. for 60 minutes in a 0.2 mL PCR tube, afterwhich 2.5 μl of the CLPA reaction was used as template in a real-timePCR reaction with 40 amplification cycles. Real-time PCR data wasaveraged for the duplicate samples and is presented in Table 3 (Ct valuecolumn). Minimal differences in Ct value between RNA and DNA targetmimics were observed indicating similar probe ligation efficiency on RNAand DNA substrates.

TABLE 3 CLPA Probe Sets. L-Probe S-Probe Target Mimic (1 nM) (1 nM) (10pM) SEQ ID SEQ ID SEQ ID Ct Sample Identifier NO NO NO value 1 MOAP-1DNA 3 4 5 19.5 2 MOAP-1 RNA 3 4 6 20 3 DDB2 DNA 10 11 12 21 4 DDB2 RNA10 11 13 21

TABLE 4 Reagent table-Example 1 1X PCR Buffer Buffer* 12.5 ul  S-Probe(1 nM) & L-Probe (1 nM) 2.5 ul each Target Mimic (100 pM) 2.5 ul Water5.0 ul Heat at 50 C. for 1 hour *1X PCR buffer is 1.5 mM MgCL2, 50 mMKCl, 10 mM Tris-HCl pH 8.3

Example 3: Direct Analysis of DDB2 RNA Transcripts in Lysis Buffer andLysed Blood

DDB2 messenger RNA (mRNA) was prepared using an in-vitro transcriptionkit from Ambion and a cDNA vector plasmid from Origene (SC122547). Theconcentration of mRNA was determined using PicoGreen RNA assay kit fromInvitrogen. The DDB2 probe sets (Table 5) were tested with differentconcentrations of DDB2 mRNA transcript spiked into either water or wholeblood. The reactions mixture components are listed in Table 5. Samples1-4 consisted of DDB2 transcript at 10 ng, 1 ng, 0.1 ng and 0.01 ng inwater, and samples 5-8 consisted of the same concentration range spikedinto whole blood. Similar reactions protocols were followed with theexception of adding Proteinase K to the blood samples so as to reduceprotein coagulation. The procedure is as follows: The reagents wereadded in the concentrations and volumes in Table 5 and Table 6.

The RNA transcripts were stable once combined with the buffer prior tothe subsequent heat step and could be stored in this buffer solution forseveral days without observing any significant degradation of the RNA.The S-probes, mRNA transcript, Guanidine hydrochloride lysis buffer andeither water (samples 1-4) or whole blood (samples 5-8) were heated to80° C. for 5 minutes and then they were moved to a 55° C. heat block.The L-probe, wash buffer, streptavidin beads and proteinase K wereadded, and the reaction was incubated at 55° C. for 60 minutes. Thesamples were removed from the heat block and the magnetic beads werecaptured using a dynal MPC 96S magnetic capture plate. The supernatantwas removed and the beads were washed 3 times with wash buffer. DyNamoSYBR green PCR master mix (25 ul, 1×) and universal primers (SEQ ID NOS1 and 2, 300 nM) were added to the beads and samples were heat cycledusing a Stratagene MX4000 realtime PCR instrument for 30 cycles (95° C.for 15 minutes, 30 cycles 95° C. (10 s), 60° C. (24 s), 72° C. (10 s)).The Ct values were recorded and the amplified samples were injected intoan Agilent Bioanalyzer 2100 so as to verify the length of the amplicons.All amplicons showed the correct size (˜96 bp) and the performance wascomparable for the blood and water samples demonstrating the ability todirectly analyze RNA in lysed blood. The results are summarizes in Table7 below. In additional experiments it was shown that the length of time(from a few minutes to several hours and even up to several days) thatthe RNA transcripts were stored in the buffer solution prior to theinitial heat step did not significantly alter the results summarized inTable 7.

TABLE 5 CLPA Probe Sets. L-Probe S-Probe Sample Identifier (1 nM) (1 nM)RNA Transcript 1-8 DDB2 SEQ ID SEQ ID Origene Plasmid NO: 10 NO: 11SC122547

TABLE 6 DDB2 reaction mixture. Samples 1-4 5-8 GuHCL Lysis Buffer (2X)12.5 μl 12.5 μl S-Probe (5 nM) 1 μl 1 μl RNA Transcript (10 ng/ul to 1μl 1 μl 0.01 ng/ul) Whole Blood 0 μl 12.5 μl Water 12.5 ul 0 μl Heat 80°C. for 5 min, chill on ice Wash Buffer 20 μl 15 μl L-Probe (5 nM) 1 μl 1μl Dynal M-270 Beads 2 μl 2 μl Proteinase K (10 mg/ml) 0 μl 5 μl Total50 μl 50 μl Incubate 55° C. for 60 min. a) GuHCL lysis buffer (1X) is 3MGUHCL, 20 mM EDTA, 5 mM DTT, 1.5% Triton, 30 mM Tris pH 7.2). b) WashBuffer is 100 mM Tris (pH 7.4), 0.01% Triton.

TABLE 7 Summary results of water versus blood Assay DDB2 Conc Ct valueSample 1 10 ng 13.5 Water 2 1 ng 17 Water 3 0.1 ng 20.2 Water 4 0.01 ng24 Water 5 10 ng 13.5 Blood 6 1 ng 16 Blood 7 0.1 ng 19.2 Blood 8 0.01ng 23.5 Blood

Example 4: 3-Probe CLPA-CE Assay

Reactions were prepared in duplicate as presented in Table 8 using DNAtarget mimic probe SEQ ID NO 24 and the 3-probe CLPA probe set (SEQ IDNOS 20, 21 and 22). The probe numbers refer to the SEQ ID NOS inTable 1. The reagents were added in the concentrations and volumes inTable 9. The S-probes, L-probe and target mimics were immediately heatedto 50° C. for 60 minutes in a 0.2 mL PCR tube, after which 2.5 μl of theCLPA reaction was used as template in a Dynamo SYBR green PCR reactionwith 25 amplification cycles. Real-time PCR data was averaged for theduplicate samples and is presented in Table 8 (Ct value column). A 1 μlsample of each reaction was then analyzed via Agilent Bioanalyzer 2100to determine the size of the reaction product.

TABLE 8 CLPA Probe Sets. 3′-S probe 2L-Probe 5′-S Probe Target MimicAmplicon Samples Identifier SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO sizeCt value 1&2 GAPDH 20 21 22 24 About 135 16.3 bp 3&4 Negative 20 21 2224 None No CT observed Probes at 1 nM concentration; target mimic at 10pM concentration.

TABLE 9 Reagent table-Example 4 1X PCR Buffer Buffer* 12.5 μl  3 and 5′S-Probe (10 nM) & 2L- 2.5 μl each Probe (10 nM) Target Mimic (1 nM) 2.5μl Water 2.5 μl Heat at 50° C. for 1 hour *1X PCR buffer is 1.5 mMMgCL2, 50 mM KCl, 10 mM Tris-HCl pH 8.3

Example 5: Multiplex Real-Time CLPA Detection of mRNA

In a 0.2 ml PCR tube were added 4 sets of CLPA reagents that wereengineered to possess unique binding sites for different color duallabeled probes. The reactions were prepared as indicated in Table 10 andTable 11. The CLPA probes sets and dual labeled probes correspond to SEQID NOS 25 through 36 in Table 1. The S and run-off transcript mRNA(GAPDH, PCNA and DDB2) were added to 2× lysis buffer (GuHCL lysis buffer(1×) is 3M GUHCL, 20 mM EDTA, 5 mM DTT, 1.5% Triton, 30 mM Tris pH 7.2).The mRNA transcripts were stable once combined with the buffer prior tothe subsequent heat step and could be stored in this buffer solution forseveral days without observing any significant degradation of the RNA.The solution was then heated to 80° C. for 5 min. The samples werecooled on ice and streptavidin coated magnetic beads (DYNAL M-270) andL-probe were added. The samples were heated at 50° C. for 1 hour. Themagnetic beads were captured on a DYNAL MPC plate and washed twice withwash buffer. The beads were recaptured and dynamo PCR 1× mastermix wasadded with the 4 different dual labeled probes and universal PCR primers(25 ul total volume). The samples were heat cycled using a StratageneMX4000 realtime PCR instrument for 30 cycles (95° C. for 15 minutes, 30cycles 95° C. (10 s), 60° C. (24 s), 72° C. (10 s)) with proper filtersfor monitoring the fluorescence in the FAM, Cal Fluor orange 560, CalFluor Red 610, and Quasar 670 channels. The Ct values observed for eachchannel were recorded and are indicated in Table 10.

TABLE 10 Multiplex reagents used in Example 5. S Probes (25 pM) L Probes(25 pM) Ct(FAM)- Ct(560)- Ct(610)- Ct(670)- Samples SEQ ID NOs SEQ IDNOs Targets GAPDH3 DDB2 PCNA GAPDH4 1 & 2 26, 29, 25, 28 250 ng yeasttRNA; 40 25.5 24.5 24.8 25.8 32, 35 31, 34 pg GAPDH(Origene SC118869),40 pg PCNA (SC118528), 40 pg DDB2 (SC122547) mRNA 3 & 4 26, 29, 25, 28250 ng yeast tRNA No Ct No Ct No Ct No Ct 32, 35 31, 34 (negative) 5 & 626, 29, 25, 28 250 ng yeast tRNA; 40 22.1 24.5 22.1 22.2 32, 35 31, 34pg GAPDH(Origene SC118869), 40 pg PCNA (SC118528), 40 pg DDB2 (SC122547)mRNA 7 & 8 26, 29, 25, 28 250 ng yeast tRNA No Ct No Ct No Ct No Ct 32,35 31, 34 (negative)

TABLE 11 Additional reagents used in Example 5. GuHCL Lysis Buffer (2X)12.5 μl S-Probes (0.25 nM Stock of each) 5 μl mRNAs (250 ng tRNA +/−mRNAs) 5 μl Water 2.5 μl Heat 80° C. for 5 min, chill on ice Water 18 μlL-Probes (0.25 nm stock of each) 5 μl Beads 2 μl Total 50 μl Incubate50° C. 1 Hour

Example 6: Formalin-Fixed, Paraffin-Embedded (FFPE) CLPA Assay

Protocol:

-   1. 25 ul CLPA Lysis Buffer (6M GuHCl, 40 mM EDTA, 10 mM DTT, 3%    Triton X-100, 100 mM Tris pH 7.5) containing 400 pM of each S probe    (Sequence IDs 47, 49, 51, 53, and 55) and 25 ul of TE buffer were    added to a 200 ul PCR tube.-   2. Formalin-Fixed, Paraffin-Embedded (FFPE) tissue blocks were    sectioned using a microtome blade (Leica, RM 2155) to a thickness of    5 microns and fixed on pathology glass slides. FFPE sample with an    approximate size of 2 mm×5 mm×5 microns were scraped off the glass    slide and added to PCR tubes.-   3. The PCR tube containing the FFPE sample and lysis buffer is    sonicated at 50 Hz in a water bath sonicator (Branson Ultrasonics)    for 5 minutes at 55° C.-   4. The tube is removed from the sonication bath and 40 ul of    Proteinase K solution (2.5 mg/ml) and 10 ul of L-probe solution (1    nM in each probe, Seq. ID 46, 48, 50, 52, and 54) were added with    mixing.-   5. The tubes were incubated at 55° C. for 3 hours.-   6. The samples were removed from the heat block, and the samples    were centrifuged for 1 min at 3000 rpm using a benchtop swing-rotor    centrifuge.-   7. Only the supernatant were transferred to a fresh PCR tube.-   8. 2.0 ul on Invitrogen/Dynal M-270 streptavidin coated magnetic    beads were added, mixed and the samples were incubated for 5    minutes.-   9. The beads were captured using a Dynal MPC 96-S magnetic plate and    the supernatant was removed.-   10. The beads were washed 2 times with 200 ul of wash buffer (100 mM    tris buffer, pH 7.0)-   11. The final wash was discarded and the beads were resuspended in    10 ul of Dynamo F-450 Taq polymerase master mix (Finnzymes) and 10    ul of PCR primer set (600 nM each of sequence ID 56 and 23).-   12. The samples were then thermally cycled according to the    protocol: 95° C. 10 min ramp; 28 cycle PCR: 94° C. for 10 seconds;    60° C. for 20 seconds; 72° C. for 20 seconds.-   13. At the thermal cycling was complete, 1 ul Exonuclease was added    to each PCR reaction tube and the samples were incubated for 20 min    at 37° C. followed by 95 C for 2 min.-   14. 2.5 ul of PCR reaction was removed from each well and added to    22.5 ul of Luminex Bead Mix. The Luminex Bead Mix was prepared as    follows. Combine 2500 microspheres for each set per reaction (Beads    66, 63, 68, 12 and 72). Dilute/concentrate the MagPlex-TAG    microsphere mixture to 111 of each microsphere set per uL in 1.1× Tm    Hybridization Buffer by vortex and sonication for approximately 20    seconds.-   15. The samples are then incubated at 95° C. for 1.5 min and then    37° C. for 1 hour with agitation using a Thermomixer.-   16. Prepare Reporter Mix by diluting SAPE to 10 ug/mL in 1× Tm    Buffer (0.2M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0).-   17. Add 100 ul Reporter Mix to each well. Mix gently.-   18. Incubate at 37° C. for 15 minutes.-   19. The MagTag assay was then run on the Luminex instrument    according to manufacturer's protocols    The results are shown in FIG. 9.

Example 7: Target Capture CLPA Blood Assay

In a 0.2 ml PCR tube, 25 ul of whole blood was mixed with 25 ul of 2×GuHCl Lysis buffer. The sample was vortexed briefly. To this tube wasadded 25 ul of a solution containing 2.0 nM target capture probes (TC)and 2.0 nM S-probe probes. The solution was mixed and heated to 80° C.for 5 min and then cooled to 55° C. While at 55° C., 50 ul of a solutioncontaining 1.0 nM L-probes and 2 mg/ml of proteinase K were added. TheS-probe, L-probe and TC probe sequences are listed in Table 1, SequenceID 37-45. The solution was mixed and heated to 55° C. for 30 minutes.After 30 minutes, 2 ul of M-270 streptavidin coated magnetic beads wereadded. The samples were mixed by gentle pipetting followed by incubationfor 5 minutes longer at 55° C. The samples were removed from the heatblood and immediately placed onto a 96-well magnetic concentrator plate(Dynal). After 15 seconds, the supernatant was removed completely. Thebeads were washed 3 times with 180 ul of wash buffer (100 mM trisbuffer, pH 7.4, 0.01% triton). The was buffer was removed and the PCRmix containing 300 nM each of primer 1 and 2 were added. The sampleswere thermal cycled for 28 cycles (95° C. 2 min, 28 cycles 94° C. (10s), 60 C (20 s) and 72° C. (20 S)). After thermal cycling, the 2.0 ul ofPCR product was mixed with 1 ul of ABI Genescan 600 V2 size standard and17 ul of formamide. The samples were heated to 95° C. for 5 minutes andthen placed on an ABI 3500 Capillary electrophoresis instrument foranalysis. Only 3 peaks were observed within the range of 110-200 basepairs on the CE trace.

TABLE 13 Example 7 results Gene Size Peak Height (RFU) MRPS5 137 30000PCNA 146 28000 CDR2 187 4000

The present specification provides a complete description of themethodologies, systems and/or structures and uses thereof in exampleaspects of the presently-described technology. Although various aspectsof this technology have been described above with a certain degree ofparticularity, or with reference to one or more individual aspects,those skilled in the art could make numerous alterations to thedisclosed aspects without departing from the spirit or scope of thetechnology hereof. Since many aspects can be made without departing fromthe spirit and scope of the presently described technology, theappropriate scope resides in the claims hereinafter appended. Otheraspects are therefore contemplated. Furthermore, it should be understoodthat any operations may be performed in any order, unless explicitlyclaimed otherwise or a specific order is inherently necessitated by theclaim language. It is intended that all matter contained in the abovedescription and shown in the accompanying drawings shall be interpretedas illustrative only of particular aspects and are not limiting to theembodiments shown. Unless otherwise clear from the context or expresslystated, any concentration values provided herein are generally given interms of admixture values or percentages without regard to anyconversion that occurs upon or following addition of the particularcomponent of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entirety for all purposes. Changes in detail or structure may bemade without departing from the basic elements of the present technologyas defined in the following claims.

What is claimed is:
 1. A method for detecting a plurality of differenttarget nucleic acids in a sample, wherein each target nucleic acidcomprises a first target domain adjacent to a second target domain and athird target capture domain located upstream or downstream from saidfirst and second target domains, said method comprising: (a) providing aplurality of ligation substrates each comprising: (i) one of said targetnucleic acids; (ii) a first set of ligation probes comprising: (A) afirst nucleic acid ligation probe comprising: (1) a first probe domainhybridized to a first target domain of said one target nucleic acidsequence; (2) a 5′-ligation moiety; and (B) a second nucleic acidligation probe comprising: (1) a second probe domain hybridized to asecond target domain of said one target nucleic acid sequence; and (2) a3′ ligation moiety; (b) ligating said first and second ligation probeswithout the use of a ligase enzyme to form a first plurality of ligationproducts; (c) hybridizing target capture probes comprising a capturemoiety to said third target domain of said target nucleic acids to formtarget complexes; (d) capturing said target complexes on a surface usingsaid capture moiety; (e) amplifying said ligation products to formamplicons; (f) detecting said amplicons, thereby detecting said targetnucleic acids.
 2. The method of claim 1, wherein: said target nucleicacids further comprise a fourth target domain adjacent to a fifth targetdomain; said plurality of ligation substrates each further comprises asecond set of ligation probes comprising a third nucleic acid ligationprobe hybridized to said fourth target domain and a fourth nucleic acidligation probe hybridized to said fifth target domain; said methodfurther comprising ligating said third and fourth ligation probes in theabsence of a ligase enzyme such that said one target nucleic acidsequence comprises multiple ligation products; and said detecting step(f) comprises detecting amplicons generated from said multiple ligationproducts.
 3. The method of claim 2, wherein said target nucleic acidsfurther comprise a sixth target domain adjacent to a seventh targetdomain; said plurality of ligation substrates each further comprises athird set of ligation probes comprising a fifth nucleic acid ligationprobe hybridized to said sixth target domain and a sixth nucleic acidligation probe hybridized to said seventh target domain; said methodfurther comprising ligating said third and fourth ligation probes in theabsence of a ligase enzyme such that said one target nucleic acidsequence comprises multiple ligation products.
 4. The method of claim 1,wherein each of said ligation probes further comprises a primersequence.
 5. The method of claim 1, wherein at least one of saidligation probes of each set of ligation probes further comprise avariable spacer sequence such that said amplicons have a target specificlength.
 6. The method of claim 1, wherein only one ligation probe ofeach set of ligation probes comprises said variable spacer sequence. 7.The method of claim 5, wherein said variable spacer sequence iscontained between said probe domain and said primer of said ligationprobe.
 8. The method of claim 1, wherein said capture moiety of saidtarget capture probe comprises a member selected from: a capture nucleicacid sequence, a bead and a binding partner of a binding partner pair.9. The method of claim 1, wherein said 5′ ligation moiety comprisesDABSYL and said 3′ ligation moiety comprises phosphorothioate.
 10. Themethod of claim 1, wherein said 3′ ligation moiety comprises DABSYL. 11.The method of claim 1, wherein said sample was collected into a buffercomprising guanidinium hydrochloride (GuHCl).
 12. The method of claim11, wherein said sample is blood.
 13. The method of claim 1, whereinsaid target nucleic acids comprise RNA.
 14. The method of claim 1,wherein said detecting step (f) utilizes a technique selected from thegroup consisting of capillary electrophoresis, mass spectrometry,microarray analysis, sequencing, real-time PCR, optical detection,fluorescence detection, bioluminescence detection, chemiluminescencedetection, electrochemical detection, electrochemiluminescence detectionand lateral flow detection.
 15. The method of claim 1, wherein saidhybridizing step (c) occurs simultaneously with said ligating step (b).16. A method for detecting a plurality of target RNA sequences in asample, wherein each target nucleic acid sequence comprises an adjacentfirst and second target domain and a third target domain, said methodcomprising: (a) collecting a sample into a buffer to form a stabilizedsample; (b) conducting an assay in accordance with claim 1 in saidstabilized sample to detect said plurality of target nucleic acids. 17.The method of claim 16, wherein said sample is stable for at least 1week at ambient room temperature.